Preparation of microgram quantities of BaP-DNA adducts using isolated rat hepatocytes in vitro.

The analysis of carcinogen-DNA adducts generally requires the preparation (by chemical or biological means) of DNA adduct standards, in amounts sufficient for chemical characterization. We have established conditions for the in vitro biological preparation of microgram quantities of DNA adducts derived from benzo[a]pyrene (BaP), fluoranthene and 7,12-dimethylbenzanthracene, using isolated rat hepatocytes. The metabolic activation of 180 microM BaP by isolated rat hepatocytes in a calf-thymus-DNA (CT-DNA)-supplemented medium resulted in the formation of 2.9 micrograms of BaP adducted to 56.7 mg of DNA. The average level of binding in this experiment was 148 +/- 8 pmol BaP bound/1 mg DNA, which compares favorably to the 10-30 pmol BAP/1 mg DNA which is typical of mouse skin adducts in vivo. In another experiment, BaP-DNA adduct formation in calf-thymus DNA added to hepatocyte incubations was further increased to 327 +/- 27 pmol/mg DNA, by physical shearing of the DNA prior to the incubation. The HPLC profile of the BaP adducts produced using hepatocytes plus CT-DNA is virtually indistinguishable from that produced by tumor-initiating doses of BaP applied to mouse skin in vivo, and the major DNA adduct formed by the hepatocytes co-elutes with the (+)-anti-diol-epoxide adduct of deoxyguanosine. Similar experiments using fluoranthene and 7,12-dimethylbenzanthracene also resulted in substantial DNA adduct formation; however, incubations using dibenz[a,h]anthracene did not. These results indicate that isolated rat hepatocytes in vitro can be useful for the preparation of DNA adducts of a number of polycyclic aromatic hydrocarbons, in quantities sufficient for chemical characterization.

Mass spectral characteristics of derivatized metabolites of benzo [A] pyrene.

The electron impact (EI) mass spectra of the permethyl, peracetyl and per(trifluoroacetyl) derivatives of hydroxylated benzo[a]pyrene (B[a]P) metabolites were determined and the fragmentation chemistry producing the spectra elucidated. The metabolites investigated were: 3-hydroxy-B[a]P; 7,8-dihydro-7,8-dihydroxy-B[a]P; 7,8,9,10-tetrahydro-7,8,9-trihydroxy-B[a]P; and 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy-B[a]P. In addition, the positive and negative methane chemical ionization spectra were determined for the derivatives of the BP-tetrol. The EI fragmentation patterns of the methylated metabolites that contained partially saturated rings were complex and, in the case of the di- and trimethoxy compounds, included apparent violations of the even-electron rule. The permethylated triol and tetrol cleaved through a retro-Diels-Alder reaction. The EI spectra of the peracetates were dominated by losses of acetic acid and ketene. The per(trifluoroacetyl) species fragmented by losing elements of trifluoroacetic acid, trifluoracetate radical and trifluoroacetyl. The spectra obtained from the permethylated tetrol permitted accurate prediction of the corresponding permethylated derivatives of tetrol metabolites of chrysene and benz[a]anthracene. The ability to predict spectra may be useful in trace analysis of hydrocarbon metabolites in biological samples.

Uptake, distribution, and elimination of dietary quinoline by rainbow trout (Salmo gairdneri).

1. Rainbow trout (Salmo gairdneri) readily absorbed 14C-quinoline from pelleted food and whole-body concentrations reached apparent equilibrium after 10 days feeding. Maximum whole body concentrations of 14C-quinoline were only 30 eta g/g of quinoline plus metabolites after 7 days depuration. 2. Initial rates of uptake and elimination varied widely among tissues. The uptake rate constants ranged from 0.0006/day for muscle to 0.1455/day for gallbladder plus bile. Mean elimination half-life for quinoline and its metabolites ranged from 0.4 days in gills to 8.7 days for muscle. 3. Depending on tissue, 58-83% of the stored radioactivity was present as metabolites. About 14% of the radioactivity in the bile was present as glucuronide conjugates. 4. Rainbow trout eliminated 66% of the ingested dose 24 hr after feeding. Biliary and fecal excretion were minor routes of elimination.