Rainbow trout (Oncorhynchus mykiss) and ultra-low dose cancer studies

Abstract

Cancer risk assessment utilizing rodents requires extrapolation across five orders of magnitude to estimate the Virtually Safe Dose (VSD). Regulatory agencies rely upon the Linear Extrapolated Dose (LED) except when sufficient information on mechanism of action justifies alternative models. Rainbow trout (Oncorhynchus mykiss) has been utilized at Oregon State University as a model for human cancer for forty years. Low cost and high capacity, made possible by our unique facility, along with low spontaneous background and high sensitivity, allow design and conduct of statistically challenging studies not possible in rodents. Utilization of custom microarrays demonstrates similarities in gene expression in trout and human hepatocellular carcinoma (HCC). We have completed one study employing over 42,000 trout with dibenzo[a,l]pyrene (DBP) and determined the dose resulting in 1 additional cancer in 5000 animals, a 50-fold enhancement over the mouse ED(01) study. Liver tumor incidence at low dose deviated significantly from linearity (concave down), whereas, DBP-DNA adductions deviated slightly (convex up). A second study is underway with aflatoxin B(1) (AFB(1)). Results to date indicate AFB(1) at low dose, in contrast to DBP, elicits a linear dose-response function on the log-log scale which falls below the LED with a slope slightly greater than 1.0. Such studies demonstrate the statistical power of the trout cancer model and strengthen the case for incorporation of these data-sets into risk assessment for these environmental human carcinogens.

Figure 1

Estimated number of animals required in each group to provide 50% power to demonstrate a statistically significant difference (p-value of 0.1) for one excess tumor in 1000 animals based on background tumor incidence. Calculations were performed according to LaMorte WW: Sample size calculations in research. Boston University Medical Center (http://www.uml.edu/ora/institutionalcompliance/IACUC/LaMorte%20Sample%20Size%20Calc%20Paper.pdf).

Figure 2

Tanks (top) housing rainbow trout (bottom) used in the cancer studies at the SARL.

Figure 3

(A) Bioactivation of DBP through CYP dependent formation of DBPDE or aldo-ketoreductase (AKR) formation of the catechol followed by (B) redox cycling.

Figure 4

ED₀₀₁ Liver tumor incidence with DBP. Each point represents the means (bars 95% confidence interval) of the four quartiles. The solid line is calculated from the LED at tumor incidences of 10% and higher. The small arrows indicate the estimated dose using the two extrapolation methods (LED versus probit fit) that would result in a 10⁻⁶ incidence.

Figure 5

Metabolic pathways for bioactivation and detoxication of AFB₁.

Figure 6

Preliminary Tumor Incidence for Quartiles 1 and 2. Background incidence (0.1%) has been subtracted.

Figure 7

Cell proliferation and apoptosis in trout liver as a function of AFB₁ dose.