J Pharmacol Toxicol Methods. 2002 Jan-Feb;47(1):25-32.  

Porphyrinogenic xenobiotic-induced N-alkylprotoporphyrin IX formation: a
bioassay utilizing chick embryo hepatic ferrochelatase.

Gamble JT, Nakatsu K, Marks GS.

Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen's
University, K7L 3N6, Kingston, Ontario, Canada.

INTRODUCTION: 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. DISCUSSION: 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.

PMID: 12387936 [PubMed - indexed for MEDLINE]