Further flow cytometric studies of the effects of triethylenemelamine on somatic and testicular tissues of the rat.

Exposure to the mutagen triethylenemelamine on rat bone marrow, blood, and testis was studied using flow cytometry of DAPI-stained nuclei. Increased coefficients of variation (CVs) of the G1 peaks were observed in bone marrow and blood after both 1 d and 5 d exposures. After 5 d exposure and 7 d recovery both tissues had recovered, in some cases to significantly lower CVs. Increased CVs of the 1C peak of testis were observed only after 5 d exposure to the high dose with no subsequently observed recovery. Bone marrow cells also were stained with Hoechst 33258 and Propidium Iodide. No differences among dyes were observed indicating that increased CVs likely are due to DNA damage resulting from interactions with the mutagen rather than differences in how the dyes bind to DNA relative to mutagen binding. This study demonstrates that differences occur among tissues in how quickly they respond and recover from mutagen exposure. Increased CVs, cell cycle alterations, and decreased CVs after recovery are all potentially useful biomarkers of effect for laboratory and field studies in environmental toxicology.

Flow-cytometric analysis of the effects of triethylenemelamine on somatic and testicular tissues of the rat.

The effects of short-term (24 h) exposure to triethylenemelamine on cellular DNA in five tissues (bone marrow, spleen, kidney, large intestine, and testis) of the rat were studied using flow cytometry. Mean coefficients of variation of the G1 peaks were increased in both the low and high dosage groups relative to controls. Bone marrow exhibited the highest degree of effect, possibly due to the rapid rate of cell division in that tissue, and spleen was next highest. Thus, hematopoietic tissues are highly responsive to short-term, acute exposure to this mutagen. The results of the flow-cytometry assay closely paralleled a simultaneous chromosomal assay conducted on bone marrow of the same rats. These data are interpreted to be consistent with the hypothesis that the observed increase in mean coefficients of variation is due to the clastogenic effects of the mutagen and subsequent unequal distribution of DNA among the daughters of affected cells.

Toxicokinetics of 1,2-dibromo-3-chloropropane (DBCP) in the rat.

The objective of this study was to determine the kinetics of absorption, distribution, and elimination of DBCP after intravenous (iv) administration in plasma, and after oral administration in water or corn oil, to conscious, fed, male Fischer 344 rats. Rats were prepared with an external jugular vein cannula and were dosed with 0.1, 1, or 10 mg/kg DBCP into the penile sinus or orally as a solution in water or in corn oil (1 mg/kg only). Blood was sampled at various times up to 12 hr, concentrations of DBCP were determined by gas chromatography, and data were evaluated by classical pharmacokinetic techniques. After oral administration in water, absorption of DBCP was rapid, and the distribution and elimination phase was biexponential. There did not appear to be any saturation of DBCP absorption, distribution, or elimination at the high oral or iv dose. After oral administration of DBCP in a corn oil vehicle, absorption was prolonged, suggesting retention of DBCP in the stomach; this could contribute to the toxic effects of DBCP on the forestomach when chronically administered in corn oil. The areas under the blood concentration/time curve were similar regardless of vehicle, suggesting that systemic toxicity might be independent of the vehicle.

Evidence that epichlorohydrin is not a toxic metabolite of 1,2-dibromo-3-chloropropane.

The major urinary metabolite of 14C-epichlorohydrin, after oral administration to rats, was identified previously (Gingell et al. 1985) to be N-acetyl-S-(3-chloro-2-hydroxypropyl)-L-cysteine (ACPC) at 36% of the administered dose. In a similar study reported here, 1,2-dibromo-3-chloropropane (DBCP) was metabolized to at least 20 radioactive urinary metabolites. ACPC was only a minor metabolite (4%) of DBCP. Epichlorohydrin was metabolized in vitro by rat liver microsomes to alpha-chlorohydrin, but DBCP was not metabolized to epichlorohydrin or alpha-chlorohydrin under similar conditions. Covalent binding of radioactivity to liver microsomal proteins occurred for both substrates, but was less for 14C-epichlorohydrin than for 14C-DBCP. Addition of 3,3,3-trichloropropylene oxide, an inhibitor of epoxide hydrolase, increased the extent of protein binding of epichlorohydrin, but decreased the amount of 14C-DBCP which was bound. The data indicate the epichlorohydrin is not a significant in vivo nor in vitro metabolite of DBCP in the rat, and is unlikely to be responsible for the toxicity of DBCP.

Disposition and metabolism of [2-14C]epichlorohydrin after oral administration to rats.

A comprehensive disposition and metabolism study of epichlorohydrin (ECH) has not been previously reported. In this study, male Fischer 344 rats were dosed (6 mg/kg) orally with [2-14C]ECH (98% radiochemically pure) as an aqueous solution and killed after 3 days. Approximately 38% of the radioactive dose was exhaled as CO2, 50% was excreted as metabolites in the urine, and 3% was present in the feces. Radioactivity in tissues accounted for the remainder of the administered dose. When expressed per gram of tissue, radioactivity was highest in liver, kidney, and forestomach. The half-life of initial elimination of radioactivity in both the urine and exhaled air was about 2 hr, indicating that ECH was rapidly absorbed and metabolized. The major metabolites in the urine were identified as N-acetyl-S-(3-chloro-2-hydroxypropyl)-L-cysteine and alpha-chlorohydrin, about 36 and 4% of the administered dose, respectively. Finding these metabolites, which have not been previously reported, is consistent with the initial metabolic reactions being conjugation of the epoxide with glutathione and hydration of the epoxide.

Transcription and hybridization of 125I-cRNA from flow sorted chromosomes.

Metaphase chromosomes from the Chinese hamster cell line M3-1 were separated by means of a flow sorter. Two chromosome fractions were used for this study: A, which consisted of 95% pure chromosome no. 1, and B, which was 90% pure chromosome no. 2. The DNA of 10(6) chromosomes of each type was purified, and a 125I-cRNA transcript was synthesized in a reaction containing E. coli RNA polymerase and carrier-free 125I-CTP (1.7 Ci/mumole). The cRNA product synthesized with template DNA from 10(5) sorted chromosomes contained more than 10(6) dpm. The electrophoretic mobility profiles of the cRNAs on 7.5% SDS acrylamide gels demonstrated that more than 50% of the ribo-polymers were equal to or longer than marker E. coli met-tRNAf. In hybridization reactions 21% and 17% of the transcripts from Chinese hamster whole cell and sorted chromosome DNA hybridized to Chinese hamster DNA and did not hybridize significantly over background in reactions containing calf DNA at Crt values of 1.3 and 1.9 x 10(2) mole sec/l. Labelled cRNAs transcribed from the DNA of sorted chromosomes hybridized with the DNA of each sorted chromosome fractions at a Crt of 0.6 mole sec/l. This study demonstrated that the DNA can be (1) recovered from small numbers of highly purified flow sorted chromosomes, (2) used as template by E. coli RNA polymerase and (3) used to prepare a cRNA in reactions containing polymerase and carrier-free 125I-CTP to yield a product which can be employed for hybridization analysis.

Simultaneous observation of quinacrine bands and silver grains on radiolabeled metaphase chromosomes.

A procedure is described for quinacrine banding of radiolabeled metaphase chromosomes for autoradiography. The chromosomes can be labeled either in vivo or by in situ hybridization. The banding procedure involves treating the slides with RNase and formamide and staining in quinacrine. The slides are then processed for autoradiography. After development of the photoemulsion, the chromosomes can be karyotyped with UV light by their fluorescent banding patterns and the silver grains overlaying the chromosomes can be demonstrated by the addition of tungsten light. It is possible by careful manipulation of the visible light to simultaneously observe both fluorescent bands and silver grains. This technique should significantly increase the accuracy of chromosome identification after autoradiography and decrease the time and effort required for such analysis.