In vitro-in silico study on the influence of dose, fraction bioactivated and endpoint used on the relative potency value of pyrrolizidine alkaloid N-oxides compared to parent pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) are phytotoxins found in food, feed and the environment. Yet, limited data exist from which the relative potency of a PA-N-oxide relative to its corresponding PA (REP_{PANO to PA}) can be defined. This study aims to investigate the influence of dose, fraction bioactivated and endpoint on the REP_{PANO to PA} of a series of pyrrolizidine N-oxides using in vitro-in silico data and physiologically based kinetic (PBK) modeling. The first endpoint used to calculate the REP_{PANO to PA} was the ratio of the area under the concentration–time curve of PA resulting from an oral dose of PA-N-oxide divided by that from an equimolar dose of PA (Method 1). The second endpoint was the ratio of the amount of pyrrole-protein adducts formed under these conditions (Method 2). REP_{PANO to PA} values appeared to decrease with increasing dose, with the decrease for Method 2 already starting at lower dose level than for Method 1. At dose levels as low as estimated daily human intakes, REP_{PANO to PA} values amounted to 0.92, 0.81, 0.78, and 0.68 for retrorsine N-oxide, seneciphylline N-oxide, riddelliine N-oxide and senecivernine N-oxide, respectively, and became independent of the dose or fraction bioactivated, because no GSH depletion, saturation of PA clearance or PA-N-oxide reduction occurs. Overall, the results demonstrate the strength of using PBK modeling in defining REP_{PANO to PA} values, thereby substantiating the use of the same approach for other PA-N-oxides for which in vivo data are lacking.