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The biomarker in this study was chromosomal puffing in the salivary glands of larval Chironomus tentans. Reduced puff size was considered an indication of decreased RNA synthesis. Larvae (third or fourth instar) were exposed to test chemicals in artificial substrate for 24 or 48 h. Chromosomes from glands of individual larva were stained with methyl green-pyronin Y. Diameters of Balbiani Rings I and II (large puffs on chromosome IV) were measured using a micrometer at 400 magnification. Experiments with benzo(a)pyrene (B(a)P) have determined a lowest observable effect level (LOEL) of 0.5 nmole; however, applied doses several orders of magnitude higher (100 nmole) did not affect puffing in approximately 40% of the larvae. The degree of response varied with the individual, and puffing was seldom inhibited completely. Actinomycin D (Act D), a known RNA synthesis inhibitor, had a LOEL of 6 nmole. Exposure to 12 nmole Act D stopped puffing completely in one hundred percent of the larvae. Therefore, this novel biomarker identified a subpopulation of individuals suspected to be tolerant to B(a)P but not Act D. Responding larvae were affected in a dose-related manner with increasing applied doses corresponding to greater reduction in puff size. Individual variation was not due to differences in uptake of H3-B(a)P equivalents or bioactivation as measured by protein bound metabolites. Interaction experiments showed that the effects of B(a)P and Act D were additive. This suggested that the compounds were inhibiting RNA synthesis through a common mode of action. Varied data found in laboratory and field studies using biomarkers could inpart be due to chemical-specific tolerance.
biomarker, chromosomal puffing, chironomus tentans, tolerance
Doctoral Candidate, Rutgers, The State University, New Brunswick, New Jersey
Associate Professor, Joint Graduate Program In Toxicology, Rutgers, The State University, New Brunswick, New Jersey