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PubMed2001

The mechanism of beta-hematin formation in acetate solution. Parallels between hemozoin formation and biomineralization processes.

Formation of beta-hematin in acidic acetate solution has been investigated using quantitative infrared spectroscopy, X-ray diffraction, and scanning and transmission electron microscopy. The process occurs via rapid precipitation of amorphous (or possibly nanocrystalline) hematin, followed by slow conversion to crystalline beta-hematin. Definitive evidence that the reaction occurs during incubation in acetate medium, rather than during the drying stage, is provided by X-ray diffraction and infrared spectroscopy of the wet material. The reaction follows a sigmoidal function indicative of a process of nucleation and growth and was modeled using the Avrami equation. Reaction rates and the dimensionality of growth (as indicated by the value of the Avrami constant) are strongly influenced by stirring rate. The reaction follows Arrhenius behavior, and there is a strong dependence of both the rate constant and the Avrami constant on acetate concentration. Acetate may act as a phase transfer catalyst, solubilizing hematin and facilitating its redeposition as beta-hematin. The pH dependence of the process indicates that only the monoprotonated species of hematin is active in forming beta-hematin. The formation of beta-hematin closely parallels many mineralization processes, and this suggests that hemozoin formation may be a unique biomineralization process. Inferences are drawn with respect to the formation of hemozoin in vivo.

Acetic Acid↗
PubMed1991

Formation of difluorothionoacetyl-protein adducts by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine metabolites: nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine.

19F NMR spectroscopy was used in conjunction with isotopic labeling to demonstrate that difluorothionoacetyl-protein adducts are formed by metabolites of the nephrotoxic cysteine conjugate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC). To determine which amino acid residues can be involved in adduct formation, the reactivity of TFEC metabolites with a variety of N-acetyl amino acids was also investigated. An N alpha-acetyl-N epsilon-(difluorothionoacetyl)lysine (DFTAL) adduct was isolated and characterized by 19F and 13C NMR spectroscopy and mass spectrometry. N alpha-Acetylhistidine and N-acetyltyrosine were found to act as nucleophilic catalysts to facilitate the formation of both the protein and DFTAL adducts. Adduct formation was greatly reduced when lysyl-modified protein was used as the substrate, indicating that lysyl residues are primary sites of adduct formation. However N alpha-acetyllysine, at concentrations of greater than 100-fold in excess compared to protein lysyl residues, was not effective in preventing binding of metabolites to protein. Therefore, nucleophilic catalysis at the surface of the protein may be an important mechanism for the binding of TFEC metabolites to specific lysyl residues in protein. TFEC metabolites were very reactive with the thiol nucleophiles glutathione and N-acetylcysteine. However, the predicted difluorodithioesters could not be isolated. Both stable difluorothioacetamide and less stable difluorodithioester protein adducts may play a role in TFEC-mediated nephrotoxicity.

Amines↗
PubMed1998

Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals.

We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the alpha-carbon peroxyl radical (ROO*) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed.

Acetyltransferases↗
PubMed1997

Mechanism of Formation of Novel Covalent Drug·DNA Interstrand Cross-Links and Monoadducts by Enediyne Antitumor Antibiotics

The potent enediyne antitumor antibiotic C1027 has been previously reported to induce novel DNA interstrand cross-links and drug monoadducts under anaerobic conditions [Xu et al. (1997) J. Am. Chem. Soc. 119, 1133-1134]. In the present study, we explored the mechanism of formation of these anaerobic DNA lesions. We found that, similar to the aerobic reaction, the diradical species of the activated drug initiates anaerobic DNA damage by abstracting hydrogen atoms from the C4', C1', and C5' positions of the A1, A2, and A3 nucleotides, respectively, in the most preferred 5'GTTA1T/5'ATA2A3C binding sequence. It is proposed that the newly generated deoxyribosyl radicals, which cannot undergo oxidation, likely add back onto the nearby unsaturated ring system of the postactivated enediyne core, inducing the formation of interstrand cross-links, connecting either A1 to A2 or A1 to A3, or drug monoadducts mainly on A2 or A3. Comparative studies with other enediynes, such as neocarzinostatin and calicheamicin gamma1I under similar reaction conditions indicate that the anaerobic reaction process is a kinetically competitive one, depending on the proximity of the drug unsaturated ring system or dioxygen to the sugar radicals and their quenching by other hydrogen sources such as solvent or thiols. It was found that C1027 mainly generates interstrand cross-links, whereas most of the anaerobic lesions produced by neocarzinostatin are drug monoadducts. Calicheamicin gamma1I was found to be less efficient in producing both lesions. The anaerobic DNA lesions induced by enediyne antitumor antibiotics may have important implications for their potent cytotoxicity in the central regions of large tumors, where relative anaerobic conditions prevail.

Journal Article↗
PubMedDate not supplied

Solid phase extraction purification of carboxylic acid products from 96-well format solution phase synthesis with DOWEX 1x8-400 formate anion exchange resin.

The anion exchange resin DOWEX 1x8-400 formate has been developed for the isolation or resin capture of carboxylic acids from solution phase reactions in a 96-well format using a batchwise solid phase extraction technique. Eleven different anion exchange resins (formate forms) were evaluated for their efficiency at scavenging aryl and aliphatic carboxylic acids from solution. The model carboxylic acids had pK(a)s ranging from 3.40 to 4.89. Exchange efficiency onto the resin was pK(a) dependent with the carboxylic acids but not with their diisopropylethylammonium salts. Exchange off of the resin also showed pK(a) dependence with the stronger acids requiring more concentrated solvent acid for exchange. DOWEX 1x8-400 formate was determined to have superior capacity and the fastest exchange rate. Solvents suitable for exchanging the acids onto the resin were CH2Cl2, methanol, and various solvent/water mixtures. Solvents suitable for exchanging the carboxylic acids off of the resin were TFA/solvent or HCO2H/solvent mixtures. The resin was found to swell best in CH2Cl2 and in polar protic solvents such as water, alcohols, and acids. Application of this technique to the crude product mixtures from an arrayed reductive amination and an arrayed Stille reaction provided product carboxylic acids in yields averaging 57% and purities averaging 89%.

Journal Article↗
PubMed1984

Quantum chemical studies of a model for peptide bond formation. 3. Role of magnesium cation in formation of amide and water from ammonia and glycine.

The SN2 reaction between glycine and ammonia molecules with magnesium cation Mg2+ as a catalyst has been studied as a model reaction for Mg(2+)-catalyzed peptide bond formation using the ab initio Hartree-Fock molecular orbital method. As in previous studies of the uncatalyzed and amine-catalyzed reactions between glycine and ammonia, two reaction mechanisms have been examined, i.e., a two-step and a concerted reaction. The stationary points of each reaction including intermediate and transition states have been identified and free energies calculated for all geometry-optimized reaction species to determine the thermodynamics and kinetics of each reaction. Substantial decreases in free energies of activation were found for both reaction mechanisms in the Mg(2+)-catalyzed amide bond formation compared with those in the uncatalyzed and amine-catalyzed amide bond formation. The catalytic effect of the Mg2+ cation is to stabilize both the transition states and intermediate, and it is attributed to the neutralization of the developing negative charge on the electrophile and formation of a conformationally flexible nonplanar five-membered chelate ring structure.

Amides↗
PubMed2004

Rate of enolate formation is not very sensitive to the hydrogen bonding ability of donors to carboxyl oxygen lone pair acceptors; a ramification of the principle of non-perfect synchronization for general-base-catalyzed enolate formation.

Two series of structures (1 and 2) possessing intramolecular hydrogen bonds to the lone-pair electrons of carbonyl oxygens have been examined to reveal the influence of the pK(a) of the hydrogen-bond donor on the rate of general-base-catalyzed enolate formation. The geometry of the hydrogen bonds is well accepted to be appropriate for intramolecular hydrogen-bond formation. Yet, as revealed by Brønsted plots, both series show very little dependence of the rate of enolate formation on the hydrogen-bond donor ability. The intramolecular hydrogen bonds give rate enhancements only on the order of 10-100-fold, and corrected Brønsted alpha-values are slightly below 0.1. The results can be understood by interpreting them in light of the Principle of Non-Perfect Synchronization. The results are consistent with the proton transfer occurring through an asynchronous transition state with the developing negative charge localized on carbon. We postulate that catalysts of enolate formation will be most effective if the binding groups are focused on stabilizing negative charge that is forming on the enolate carbon rather than on the enolate oxygen.

Alcohols↗
PubMed2004

Autocatalytic formation of green heme: evidence for H2O2-dependent formation of a covalent methionine-heme linkage in ascorbate peroxidase.

The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the Cbeta of the 2-vinyl group to a methionine residue. To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant). The S160M variant is isolated from Escherichia coli as apo-protein. Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (kcat = 156 +/- 7 s(-1), KM = 102 +/- 15 microM) properties that are analogous to those of rpAPX. The reaction of S160M(R) with H2O2 gives a green protein (S160M(G)). Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160M(G). Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate. The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation. A mechanism that is consistent with the data is presented.

Amino Acid Sequence↗
PubMed2006

Nanosphere and nanonetwork formations of [60]fullerene-end-capped stereoregular poly(methyl methacrylate)s through stereocomplex formation combined with self-assembly of the fullerenes.

We report a novel and versatile method for constructing a supramolecular nanosphere and nanonetwork based on isotactic and syndiotactic C60-end-capped poly(methyl methacrylate)s (it- and st-PMMA-C60's) through their stereocomplex formation combined with self-assembly of the terminal C60. The stereoregular PMMA-C60's with a precisely controlled structure including molecular weight, its distribution, tacticity, and the chain-end structure were synthesized by the stereospecific anionic living polymerizations of methyl methacrylate followed by end-capping with C60, and their structures were proven by size exclusion chromatography, NMR, UV-vis, and MALDI-TOF-MS analyses. The stereoregular PMMA-C60's self-assembled to form a core-shell aggregate with C60 as the core and the PMMA chains as the shell in H2O/CH3CN (1/9, v/v) due to the solvophobic interaction of the C60 units. These it- and st-PMMA-C60 aggregates further supramolecularly assembled through iterative stereocomplex formation into nanonetworks in which the self-assembled C60 clusters were robustly connected with two- and three-dimensional arrangements. In addition, when the it- and st-PMMA-C60's were simultaneously mixed, self-assembly of the C60 units and stereocomplex formation of the it- and st-PMMA chains took place at once, resulting in the formation of uniformly sized spherical nanoparticles with resistance to heat. Similar nanonetwork architectures can be produced using it-PMMA-C60 clusters and st-PMMA prepolymers as the binder.

Journal Article↗
PubMed1984

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. 8. Stereoselectivity in metabolic intermediary complex formation with a series of chiral 2-substituted 1-phenyl-2-aminoethanes.

The formation of cytochrome P-450 metabolic intermediary (MI) complexes from the enantiomers of four 2-alkyl-substituted 1-phenyl-2-aminoethanes was investigated during reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent metabolism in liver microsomes from phenobarbital-pretreated rats. The 2-alkyl substituents were methyl (amphetamine), ethyl, n-propyl, and n-butyl groups. The chiral amines were prepared from the corresponding alkyl benzyl ketones by asymmetric hydrogenolytic transamination. Circular dichroism analysis showed that all the amines possessed the S-(+) and R-(-) configuration. The maximal velocity (Vmax(obsd) ) of complex formation increased with increasing size of the alkyl group, and for each series of enantiomers a good correlation was obtained between log Vmax(obsd) and the logarithm of the octanol/buffer partition coefficient of the substrates. With increasing lipophilicity, the S-(+) enantiomers became more active than the R-(-) isomers in generating the complex. The rates of complex formation for the faster S-(+) enantiomers coincided with those of the previously investigated racemates, indicating that the R-(-) enantiomers do not act as competitive enzyme inhibitors in the rat liver preparations. In agreement with two previous studies, the results from the present investigation establish a stereoselectivity in cytochrome P-450 MI complex formation by 1-phenyl-2-aminoethanes. However, detection of such differences are dependent on the intrinsic activity of the compound.

Animals↗
PubMed1998

Catalysis of dialanine formation by glycine in the salt-induced peptide formation reaction.

Mutual catalysis of amino acids in the salt-induced peptide formation (SIPF) reaction is demonstrated for the case of glycine/alanine. The presence of glycine enhances dialanine formation by a factor up to 50 and enables dialanine formation at much lower alanine concentrations. The actual amounts of glycine play an important role for this catalytic effect, the optimal glycine concentration is 1/8 of the alanine concentration. The mechanism appears to be based on the formation of the intermediate Gly-Ala-Ala tripeptide, connected to one coordination site of copper(II) ion, and subsequent hydrolysis to dialanine and glycine.

Alanine↗
PubMed2004

Downregulation of osteopontin and bone sialoprotein II is related to reduced colony formation and metastasis formation of MDA-MB-231 human breast cancer cells.

Osteopontin (OPN), bone sialoprotein (BSPII), and osteonectin (ON) belong to a family of glycoproteins, which have been linked to cancer metastasis and progression. Here, we report on the selection of antisense oligonucleotides (ASOs), which are effective in reducing their protein levels. In human MDA-MB-231 breast cancer cells, the maximum inhibition of protein expression ranged from 84% (OPN) to 75% (BSPII) and 70% (ON). Erucylphospho-NNN-trimethylpropanolamine (ErPC3) was used as positive control and combination partner. Exposure to ErPC3 inhibited colony formation of MDA-MB-231 cells by 11% (10 microM), 45% (14 microM) and 78% (20 microM). The clonogenicity of breast cancer cells was reduced by 15%, 11%, 8% (5 microM), 39%, 19%, 14% (10 microM) and 46%, 39%, 21% (20 microM) in response to ASO-OPN-04, ASO-BSPII-06 and ASO-ON-03, respectively. Combination of ErPC3 with the ASOs caused additive combination effects. Pre-exposure to the ASOs, but not to the NSO, inhibited formation of osteolytic metastasis in three of four (ASO-OPN-04, P<0.03) and two of four (ASO-BSPII-06) nude rats, and reduced metastasis lesions significantly (T/C%=4.3 and 9.1, P=0.05, respectively). We conclude that downregulation of OPN and BSPII reduces colony formation of MDA-MB-231 cells and formation of osteolytic metastasis in nude rats.

Animals↗
PubMed1993

Flower-bud formation in explants of photoperiodic and day-neutral Nicotiana biotypes and its bearing on the regulation of flower formation.

The capacity to form flower buds in thin-layer explants was studied in flowering plants of several species, cultivars, and lines of Nicotiana differing in their response to photoperiod. This capacity was found in all biotypes examined and could extend into sepals and corolla. It varied greatly, depending on genotype, source tissue and its developmental stage, and composition of the culture medium, particularly the levels of glucose, auxin, and cytokinin. It was greatest in the two day-neutral plants examined, Samsun tobacco and Nicotiana rustica, where it extended from the inflorescence region down the vegetative stem, in a basipetally decreasing gradient; it was least in the two qualitative photoperiodic plants studied, the long-day plant Nicotiana silvestris and the short-day plant Maryland Mammoth tobacco, the quantitative long-day plant Nicotiana alata and the quantitative short-day plant Nicotiana otophora line 38-G-81, where it was limited to the pedicels (and, in some cases, the sepals). Regardless of the photoperiodic response of the source plants, the response was the same in explants cultured under long and short days. The finding that capacity to form flower buds in explants is present in all Nicotiana biotypes studied supports the idea that it is regulated by the same mechanism(s), regardless of the plant's photoperiodic character. However, the source plants were all in the flowering stage, and no flower-bud formation can be obtained in explants from strictly vegetative Nicotiana plants. Hence, flower formation in the explants is not identical with de novo flower formation in a hitherto vegetative plant: it is rather the expression of a floral state already established in the plant, although it can vary widely in extent and spatial distribution. Culture conditions that permit flower-bud formation in an explant are conditions that maintain the floral state and encourage its expression; conditions under which no flower buds are formed reduce this state and/or prevent its expression.

Journal Article↗
PubMed2003

Amyloid-like fibril formation in an all beta-barrel protein. Partially structured intermediate state(s) is a precursor for fibril formation.

Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a approximately 15-kDa, all beta-sheet protein devoid of disulfide bonds. In the present study, we investigate the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt acidic fibroblast growth factor (nFGF-1). The protein aggregates maximally in 10% (v/v) TFE. Congo red and thioflavin T binding experiments suggest that the aggregates induced by TFE have properties resembling the amyloid fibrils. Transmission electron microscopy and x-ray fiber diffraction data show that the fibrils (induced by TFE) are straight, unbranched, and have a cross-beta structure with an average diameter of 10-15 A. Preformed fibrils (induced by TFE) of nFGF-1 are observed to seed amyloid-like fibril formation in solutions containing the protein (nFGF-1) in the native beta-barrel conformation. Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding, multidimensional NMR, and Fourier transformed infrared spectroscopy data reveal that formation of a partially structured intermediate state(s) precedes the onset of the fibrillation process. The native beta-barrel structure of nFGF-1 appears to be disrupted in the partially structured intermediate state(s). The protein in the partially structured intermediate state(s) is found to be "sticky" with a solvent-exposed non-polar surface(s). Amyloid fibril formation appears to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and intermolecular beta-sheet formation among the extended, linear beta-strands in the protein.

Amyloid beta-Peptides↗
PubMed1985

Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides.

Cytokeratins are expressed in different types of epithelial cells in certain combinations of polypeptides of the acidic (type I) and basic (type II) subfamilies, showing "expression pairs." We have examined in vitro the ability of purified and denatured cytokeratin polypeptides of human, bovine, and rat origin to form the characteristic heterotypic subunit complexes, as determined by various electrophoretic techniques and chemical cross-linking, and, subsequently, intermediate-sized filaments (IFs), as shown by electron microscopy. We have found that all of the diverse type I cytokeratin polypeptides examined can form complexes and IFs when allowed to react with equimolar amounts of any of the type II polypeptides. Examples of successful subunit complex and IF formation in vitro include combinations of polypeptides that have never been found to occur in the same cell type in vivo, such as between epidermal cytokeratins and those from simple epithelia, and also heterologous combinations between cytokeratins from different species. The reconstituted complexes and IFs show stability properties, as determined by gradual "melting" and reassociation, that are similar to those of comparable native combinations or characteristic for the specific new pair combination. The results show that cytokeratin complex and IF formation in vitro requires the pairing of one representative of each the type I and type II subfamilies into the heterotypic tetramer but that there is no structural incompatibility between any of the members of the two subfamilies. These findings suggest that the co-expression of specific pair combinations observed in vivo has other reasons than general structural requirements for IF formation and probably rather reflects the selection of certain regulatory programs of expression during cell differentiation. Moreover, the fact that certain cytokeratin polypeptide pairs that readily form complexes in vitro and coexist in the same cells in vivo nevertheless show preferential, if not exclusive, partner relationships in the living cell points to the importance of differences of stabilities among cytokeratin complexes and/or the existence of extracytokeratinous factors involved in the specific formation of certain cytokeratin pairs.

Animals↗
PubMed1968

Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation.

Muscle cells, cultured for 1-28 days from 11-day chick embryo breast muscles, often show elaborate, three-dimensional networks of a membranous system. The network consists of tubular units which are quite regularly arranged. The tubular units composing the network are accessible to ferritin particles suspended in the culture medium; this suggests continuity with the extracellular fluid. These networks can be regarded as a special morphological elaboration of the T-system tubules. Such network formations can be seen much more often in well-developed myotubes. The networks usually exhibit a hexagonal pattern, which is formed of tubular units of a constant diameter. However, some early myotubes contain tetragonal networks, which are composed of spherical pockets with channels of lesser diameter connecting the pockets. Networks are also observed which probably represent a transitional form between these two patterns. Myotubes show many inpocketings of the sarcolemma similar to what are commonly referred to as caveolae or micropinocytotic vesicles. The similarity in configuration and dimension of the tubular units of the network to the caveolae leads to the plausible suggestion that repeated caveola-formation from the sarcolemma or T-system tubule may result in formation of these networks. In this connection, a possible mechanism of the T-system tubule formation is discussed.

Animals↗
PubMed1959

Chemical and morphological studies of bacterial spore formation. II. Spore and parasporal protein formation in Bacillus cereus var. alesti.

The development of both the spore and parasporal protein crystal of Bacillus cereus var. alesti was followed using chemical and cytological techniques. The changes which led to the formation of the fore-spore were similar to those already described for Bacillus cereus. However, adjacent to the developing fore-spore a small inclusion became discernible in phase contrast. This protein inclusion during its growth was differentiated from the chromatin and lipid-containing inclusions by sequential staining techniques. During spore and crystal formation no net synthesis of either nucleic acid was detected. Tracer studies with radioactive phosphorus confirmed that the spore chromatin was derived from that in the vegetative cell. These same studies also indicated that a turnover of ribonucleic acid occurred during the sporulation process. During their formation both the spore and crystal incorporated methionine-(35)S from the medium and from cellular material into a bound form. Sequential extractions with alkali and with alkaline-thioglycollate reagent revealed that the solubility characteristics of the mature crystal were possibly related to the presence of intermolecular disulphide bonds which developed after the major synthesis of the crystal was complete. The synthetic nature of sporogenesis and crystal formation is discussed with reference to the concept of "endotrophic" sporulation.

Bacillus cereus↗
PubMed1996

DNA adduct formation, cell proliferation and aberrant crypt focus formation induced by PhIP in male and female rat colon with relevance to carcinogenesis.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces colon tumors in male, but not female, F344 rats. We investigated the mechanisms leading to this difference by measuring the level of PhIP-DNA adducts, the enhancement of cell proliferation and aberrant crypt focus (ACF) formation in colon mucosa. PhIP was administered in the diet at a level of 0.04% to both male and female F344 rats for 1-8 weeks. The level of DNA adducts in the colon mucosa was measured using the 32P-postlabeling method. Four major PhIP-DNA adducts were detected in fairly constant proportions in all the animals examined. The level of PhIP-DNA adducts in male and female rats was the same, indicating no direct correlation between adduct levels and carcinogenesis. Labeling indices (LIs) were determined by measuring BrdU incorporation in rats after feeding with a PhIP diet for 4, 8 and 12 weeks. After 8 weeks administration the LI had increased 1.5-fold in the colon of the male rats, but no increase was observed in the female rats. ACF formation was examined after feeding with a PhIP diet for 14 weeks. The number of aberrant crypt foci was 6.6 +/- 1.5 per rat in males and 1.9 +/- 0.5 per rat in females. Thus differences in colon tumor development in male and female rats takes place at an early stage(s). Our results suggest that, in addition to DNA adduct formation, enhanced proliferation contributes to the formation of ACFs, which are premalignant lesions of the colon.

Animals↗