Diethylnitrosamine exposure-responses for DNA ethylation, hepatocellular proliferation, and initiation of carcinogenesis in rat liver display non-linearities and thresholds

In previous exposure-response studies, we have documented non-linearities f or some of the early effects in rat liver of diethylnitrosamine (DEN) and a near no-effect levels for initiation of promotable liver neoplasms at the lowest cumulative exposure of 0.5 mmol/kg body weight; this in spite of for mation of DNA adducts and induction of hepatocellular altered foci (HAF). T o extend these investigations, in an initiation segment, young male F344 ra ts were administered four exposures of DEN ranging from a cumulative total of 0.25 mmol, which is half of the previously used low exposure, up to 2 mm ol per kg body weight, an effective initiating exposure. These exposures we re achieved by once weekly intragastric instillations of one-tenth the tota l exposures for up to 10 weeks. The initiation segment was followed by a 4 week recovery segment, to allow for remission of acute and subchronic effec ts of DEN, after which the groups were maintained on 0.06% phenobarbital in the diet for 24 weeks to promote liver tumor development in order to asses s initiation. During and after initiation and at the end of recovery, selec ted groups were studied for several crucial effects involved in hepatocarci nogenicity. The low exposure produced a low-level of DNA ethylation at both 5 and 10 weeks of exposure, measured as O-4-ethylthymidine, the most persi stent promutagenic ethylation product. At the 5 week interval, the adduct v alues of the higher exposures were less than proportional to the increment of exposure, suggestive of nonlinearity. Assessment of cellular proliferati on by staining for proliferating cell nuclear antigen revealed that the low est exposure did not increase the replicating fraction of hepatocytes durin g the initiation (10 weeks) or recovery (4 weeks) segments, whereas in the three higher exposure groups, proliferation was increased in relation to do se and time. Preneoplastic HAF expressing glutathione S-transferase-placent al-type were present at low multiplicity in control livers and their multip licity was increased in all exposure groups by the end of exposure, at whic h time the increase in the high exposure group was disproportionately great er than the increment of exposure. After phenobarbital administration in th e promotion segment, all exposure groups exhibited further HBF increases at 39 weeks. At the end of the promotion segment, no hepatocellular neoplasm was found in 80 controls or in 40 rats in the low exposure group. In the mi d-low exposure group, which was the previously studied low exposure, only o ne adenoma was found, yielding a 3% incidence, while in the two higher expo sure groups, 32 and 80% of rats exhibited liver neoplasms, which were incre ased disproportionately greater than the increments of exposure. Thus, the findings document non-linearities of early DEN effects and at the lowest cu mulative dose, a no-effect level (NEL) or threshold for initiation of promo table liver neoplasms. These findings provide a conceptual basis for unders tanding why low-level exposures to DNA-reactive carcinogens may convey no c ancer risk.