Porphyrinogenic xenobiotic-induced N-alkylprotoporphyrin IX formation: a bioassay utilizing chick embryo hepatic ferrochelatase.
BACKGROUND
The porphyrinogenicity of some xenobiotics results from mechanism-based inactivation of selected cytochrome P450 (CYP) enzymes accompanied by conversion of prosthetic heme groups to N-alkylprotoporphyrins (N-alkylPPs), some of which inhibit ferrochelatase (FC). Problems have arisen in extrapolating xenobiotic porphyrinogenicity observed in test animals to humans, due in part to differences among CYP enzymes. Our goal was to develop a bioassay to detect N-alkylPPs formed following interaction of porphyrinogenic xenobiotics with rat liver microsomal CYP.


METHODS
Seventeen-day-old chick embryo livers were homogenized, and the mitochondrial fraction was isolated. The FC activity of this fraction was determined by means of the pyridine hemochromogen method. Inhibition of FC was used to detect N-alkylPP formation following interaction of porphyrinogenic xenobiotics with rat liver microsomes.


RESULTS
The 17-day-old chick embryo hepatic mitochondrial preparation served as a stable source of FC activity, which was linear with respect to time and protein concentration. FC activity was higher than previously reported in a homogenate of 17-day-old chick embryo hepatocytes in culture and in an aqueous extract of 17-day-old chick embryo mitochondria. The EC(50) of N-methylprotoporphyrin IX in the chick embryo liver mitochondrial preparation was similar to that in the homogenate of chick embryo liver cell culture. The FC bioassay could detect N-alkylPPs formed following the interaction of porphyrinogenic xenobiotics with rat liver microsomes containing 2.4-9.0 nmol of CYP.


CONCLUSIONS
In future studies investigating N-alkylPP formation following interaction of xenobiotics with CYP enzymes, we recommend using a combination of a fluorescence technique and the chick embryo hepatic mitochondrial FC assay. This would provide information both on the formation of N-alkylPPs and distinguish between those N-alkylPPs that produced porphyrin accumulation via FC inhibition and those that do not.
Gamble
Jeremy T
JT
Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen's University, K7L 3N6, Kingston, Ontario, Canada.
Nakatsu
Kanji
K
Marks
Gerald S
GS
D000818:Animals; D001681:Biological Assay; D002642:Chick Embryo; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004790:Enzyme Induction; D005294:Ferrochelatase / Q000096:biosynthesis*; D008099:Liver / Q000187:drug effects / Q000196:embryology / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology; D011164:Porphyrias / Q000139:chemically induced / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D015262:Xenobiotics / Q000378:metabolism / Q000494:pharmacology
D011524:Protoporphyrins; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; C028025:protoporphyrin IX; D005294:Ferrochelatase