A new mutagenic compound has been isolated from ground beef which was fried at 300 degrees C for 5.5 min on each side. The new mutagen was purified using an aqueous acid extraction, XAD-2 adsorption-solvent elution, a series of preparative and analytical h.p.l.c. purification steps, and monitored with the Ames/Salmonella assay. This study reveals a new mutagen member of the amino-imidazoazaarene class of aromatic amines, having a mol. w of 224, and a formula of C13H12N4 as determined by high-resolution mass spectrometry. N.m.r. spectrometry supports the structure, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), for the new mutagen. The 1-methyl and 3-methyl synthesized isomers of PhIP were compared to the purified mutagen. The two isomers had identical mass spectra to the purified compound, but only the 1-methyl isomer showed similar u.v. and n.m.r. spectra. The two synthetic isomers were separable by h.p.l.c. and the beef derived component co-eluted with the 1-methyl-PhIP isomer. PhIP has a specific activity in the Ames/Salmonella assay of 1950 revertants/microgram. Although it is not as mutagenic as other compounds isolated from fried beef (e.g. MeIQx, 58 000 revertants/microgram) it is the most abundant mutagenic compound by mass in fried beef. PhIP is present at approximately 15 p.p.b. of the original weight of uncooked beef (accounting for 75% of the mass of genotoxic material) and contributes 18% of the total mutagenicity of the fried beef.

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Isotropic contact interaction shifts in the proton NMR spectra of pyridine-type molecules coordinated with paramagnetic nickel (II) and cobalt (II) acetylacetonates have been observed. The role of π- and σ- bonding frameworks in the transfer of unpaired electron spin density from the metal ion to ligand protons is considered. Pseudocontact interactions appear to be significant in the cobalt systems but not in the nickel systems. When the pseudocontact contribution to isotropic shifts in cobalt systems is factored out there remain contact shifts which are in good agreement with the observed nickel shifts. It is concluded that the unpaired electrons in metal eg orbitals of both cobalt and nickel systems cause contact shifts in the pyridine-type ligands whereas the t2g electron in the cobalt system does not. The t2g electron gives isotropic shifts by contributing to anisotropy in the electronic g factor.

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The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), a heterocyclic amine carcinogen detected in cooked meats, was investigated in mice. In 3-methylcholanthrene-induced mice administered 0.1, 1.0 and 10 mg/kg [14C]PhIP (i.p.), urinary and fecal excretion over 24 h accounted for 16% and 42-56% of the dose respectively. Urinary excretion of unchanged parent compound accounted for only 0.5-0.8% of the administered dose. At all doses, the major urinary metabolite was identified as 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate and this metabolite comprised approximately 5% of the dose. Uninduced mice excreted greater than 13% of a 10 mg/kg dose as the sulfate conjugate. Urinary excretion of both 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) and a glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (N-hydroxy-PhIP) was also higher (4-fold) in uninduced versus induced mice. The decreased urinary excretion of P450-derived metabolites via induction contrasted with increased metabolite formation by hepatic microsomal preparations. 4'-Hydroxy-PhIP and N-hydroxy-PhIP were produced in amounts nearly 7- and 3-fold higher respectively by induced versus uninduced microsomal incubations at 50 microM [3H]PhIP. At concentrations less than 10 microM, PhIP was almost exclusively converted by the induced preparations to an unidentified metabolite that was not retained by the C18 column. This metabolite, which also was formed in incubations with either 4'-hydroxy-PhIP or N-hydroxy-PhIP, was produced by microsomes from uninduced animals at a much slower rate. Covalent binding to microsomal protein in incubations with [3H]PhIP was concentration-dependent and 2- to 4-fold higher in induced than uninduced preparations. Covalent binding in liver and kidney of induced mice administered [14C]PhIP was dose dependent. At 10 mg/kg PhIP, adducts were produced at 1.7-fold higher levels in livers of induced versus uninduced mice, but renal binding was higher in uninduced animals. These studies indicate the importance of cytochrome P450 and other xenobiotic enzymes in the metabolism, disposition and activation of PhIP.