Cytosolic enzymes from rat tissues that activate the cooked meat mutagen metabolite N-Hydroxyamino-1-methyl-6- phenylimidazo[4,5-b]pyridine (N-OH-PhIP).

Heterocyclic amines are formed during the cooking of foods rich in protein and can be metabolically converted into cytotoxic and mutagenic compounds. These "cooked-food mutagens" constitute a potential health hazard because DNA damage arising from dietary exposure to heterocyclic amines can modify cell genomes and thereby affect future organ function. To determine enzymes responsible for heterocyclic amine processing in mammalian tissues, we performed studies to measure genotoxic activation of the N-hydroxy form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) --a common dietary mutagen. O-Acetyltransferase, sulfotransferase, kinase, and amino-acyl synthetase activities were assayed using substrate-specific reactions and cytosolic enzymes from newborn and adult rat heart, liver, spleen, kidney, brain, lung, and skeletal muscle. The resultant enzyme-specific DNA adduct formation was quantified via (32)P-postlabeling techniques. In biochemical assays with rat tissue cytosolic proteins, O-acetyltransferases were the enzymes most responsible for N-hydroxy-PhIP (N-OH-PhIP) activation. Compared to O-acetyltransferase activation, there was significantly less kinase activity and even lesser amounts of sulfotransferase activity. Proyl-tRNA synthetase activation of N-OH-PhIP was not detected. Comparing newborn rat tissues, the highest level of O-acetyltransferase mutagen activation was observed for neonatal heart tissue with activities ranked in the order of heart > kidney > lung > liver > skeletal muscle > brain > spleen. Enzymes from cultured neonatal myocytes displayed high O-acetyltransferase activities, similar to that observed for whole newborn heart. This tissue specificity suggests that neonatal cardiac myocytes might be at greater risk for damage from dietary heterocyclic amine mutagens than some other cell types. However, cytosolic enzymes from adult rat tissues exhibited a different O-acetyltransferase activation profile, such that liver > muscle > spleen > kidney > lung > brain > heart. These results demonstrated that enzymes involved in catalyzing PhIP-DNA adduct formation varied substantially in activity between tissues and in some tissues, changed significantly during development and aging. The results further suggest that O-acetyltransferases are the primary activators of N-OH-PhIP in rat tissues.

Bioactivation of the proximal food mutagen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) to DNA-binding species by human mammary gland enzymes.

We have investigated phase II activation of the food-derived mutagen 2-hydroxyamino-1-methyl-6-phenyl[4,5-b]pyridine (N-OH-PhIP) by cytosolic acetyltransferase, sulfotransferase, and tRNA synthetase/kinase enzymes from human breast tissue. Cytosol from homogenates of mammary gland tissue obtained from breast-reduction surgery or mastectomy was incubated with and without enzyme-specific cofactors, and mutagen binding of calf thymus DNA was quantified by 32P-postlabeling. In addition, microsomal fractions of mammary epithelial cells from some individuals were examined for prostaglandin H synthetase activation of N-OH-PhIP. Our results show that all four enzymes can participate in activating N-OH-PhIP, thus inducing PhIP-DNA adduct formation in human mammary cells. However, not all individuals exhibited all these activities; instead each individual showed a combination of one or more activation pathways. The present findings demonstrate that the human mammary gland has the capacity to metabolically activate a dietary mutagen by several enzyme systems, including acetyltransferase, sulfotransferase, tRNA synthetase/kinase, and prostaglandin hydroperoxidase catalysis.

Identification of an S100 target protein: glycogen phosphorylase.

An S100 binding protein from skeletal muscle, R95 000, has been purified, identified as glycogen phosphorylase, and shown to be regulated in vitro by the S100 alpha isoform. When a soluble skeletal muscle fraction was subjected to a standard purification procedure for glycogen phosphorylase, R95 000 copurified with the 95 000 molecular weight glycogen phosphorylase protein standard on SDS-polyacrylamide gels, as well as having glycogen phosphorylase activity. In addition, purified glycogen phosphorylase a and b interacted with both S100 isoforms, S100 alpha and S100 beta, by gel overlay and affinity chromatography. While S100 beta had no effect on the enzymatic activity of glycogen phosphorylase a, S100 alpha inhibited the enzymatic activity of glycogen phosphorylase a in a calcium-independent manner. Altogether, these data suggest that glycogen phosphorylase may be an intracellular S100 alpha target in skeletal muscle fibers. Furthermore, these results suggest that the inhibition of glycogen phosphorylase a activity may be responsible for the lack of fatigability of slow-twitch fibers, which express S100 alpha, when compared to fast-twitch fibers, which do not express S100 proteins.