Validation of a flow cytometric in vitro DNA repair (UDS) assay in rat hepatocytes.

An in vitro flow cytometric (FCM) DNA repair assay has been developed and validated by comparison to conventional autoradiography (ARG). Both assays measure unscheduled DNA synthesis (UDS). Cultures of hepatocytes from young male Sprague-Dawley rats were exposed to a battery of 26 chemicals plus bromodeoxyuridine (BrdUrd) or 3H-thymidine (3H-dT) for 18-20 h before harvest. Selection of test chemicals was based upon both their genotoxicity classifications and carcinogenicity bioassay results in male rats. DNA repair in chemically treated cultures was detected flow cytometrically by measuring the uptake of BrdUrd in non-replicating (G1, G2, mitotic and 4C) cells. Intracellular levels of incorporated BrdUrd were visualized by immunochemical labeling with fluorescein isothiocyanate (FITC), and total cellular DNA content was simultaneously estimated by counterstaining samples with the nucleic acid intercalator, propidium iodide (PI). Information was obtained from 10(4) cells/sample. Since repairing cells incorporate significantly less BrdUrd per unit of time than replicating cells, low intensity BrdUrd-FITC fluorescent signals from repairing cells are readily discriminated from high intensity signals from replicating cells when displayed on linear univariate histograms. Further distinction between repairing and replicating cells was achieved by displaying the DNA contents of all cells on linear bivariate histograms. Thus, repairing cells were resolved without subjecting these cultures to agents which suppress replicative synthesis (e.g., hydroxyurea). Results from these concurrent FCM and ARG investigations include the following: (1) conclusions (DNA repair positive or negative) were in agreement, with one exception, cinnamyl anthranilate, for which cytotoxic doses produced a positive FCM response, but lack of intact hepatocytes in parallel ARG preparations prevented analysis; (2) similar sensitivities for most of the positive chemicals were reported; (3) a high correlation (85%) exists between the reported genotoxicity classification and these DNA repair results in the absence of overt cytotoxicity; (4) a poor correlation exists between these DNA repair results and hepatocarcinogenesis (only 4/11 liver carcinogens tested positive) or overall carcinogenesis in the male rat (only 9/21 carcinogens tested positive). This FCM assay provides a rapid, sensitive, safe and reliable means of identifying agents which induce DNA repair in mammalian cells.

A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog.

The microsomal O-deethylation of a novel coumarin analog, 7-ethoxy-4-trifluoromethylcoumarin (EFC), to a fluorescent product was characterized. Results indicate that this analog provides a rapid, convenient and highly sensitive means to assay cytochrome P-450-mediated metabolism. Like microsomal 7-ethoxycoumarin (7-EC) O-deethylation, EFC O-deethylation responded to both phenobarbital was greater than that seen with 7-EC (5- to 6-fold over control after 50 mg/kg/day for 4 days in Sprague-Dawley rats compared to approximately 2-fold for 7-EC). Since the reaction was monitored by direct fluorometry of the product, any departures from linearity under a particular set of reaction conditions (e.g. with highly induced samples) were immediately apparent. In the absence of an NADPH-regenerating system, background drift was very low (less than 0.01 fluorescent units), so the sensitivity of the assay was limited primarily by that of the fluorometer employed. This makes the assay particularly useful in situations where test material is limited, e.g. when measuring activity in cultured hepatocytes. Its simplicity, reproducibility, and response to a variety of inducing agents also make it suitable for a rapid screening assay for cytochrome P-450 induction.

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential.

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.