The discovery of BMS-457, a potent and selective CCR1 antagonist.

A series of compounds which exhibited good human CCR1 binding and functional potency was modified resulting in the discovery of a novel series of high affinity, functionally potent antagonists of the CCR1 receptor. Issues of PXR activity, ion-channel potency, and poor metabolic stability were addressed by the addition of a hydroxyl group to an otherwise lipophilic area in the molecule resulting in the discovery of preclinical candidate BMS-457 for the treatment of rheumatoid arthritis.

Restoration of the growth suppression function of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain.

We demonstrate here that synthetic 22-mer peptide 46, corresponding to the carboxy-terminal amino acid residues 361-382 of p53, can activate specific DNA binding of wild-type p53 in vitro and can restore the transcriptional transactivating function of at least some mutant p53 proteins in living cells. Introduction of peptide 46 in Saos-2 cells carrying a Tet-regulatable His-273 mutant p53 construct caused growth inhibition and apoptosis in the presence of mutant p53 but not in its absence, confirming that the effect of the peptide is mediated by reactivation of mutant p53. Moreover, peptide 46 caused apoptosis in mutant as well as wild-type p53-carrying human tumor cell lines of different origin, whereas p53 null tumor cells were not affected. These findings raise possibilities for developing drugs that restore the tumor suppressor function of mutant p53 proteins, thus selectively eliminating tumor cells.

Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells.

Cultured bronchial epithelial and fibroblastic cells from humans were used to study DNA damage and toxicity caused by formaldehyde. Formaldehyde caused the formation of cross-links between DNA and proteins, caused single-strand breaks in DNA, and inhibited the resealing of single-strand breaks produced by ionizing radiation. Formaldehyde also inhibited the unscheduled DNA synthesis that occurs after exposure of cells to ultraviolet irradiation or to benzo[a]pyrene diolexpoxide but at doses substantially higher than those required to inhibit the resealing of x-ray-induced single-strand breaks. Therefore, formaldehyde could exert its mutagenic and carcinogenic effects by both damaging DNA and inhibiting DNA repair.

Genotoxicity of formaldehyde in cultured human bronchial fibroblasts.

Formaldehyde, a common environmental pollutant, inhibits repair of O6-methylguanine and potentiates the mutagenicity of an alkylating agent, N-methyl-N-nitrosourea, in normal human fibroblasts. Because formaldehyde alone also causes mutations in human cells, the compound may cause genotoxicity by a dual mechanism of directly damaging DNA and inhibiting repair of mutagenic and carcinogenic DNA lesions caused by other chemical and physical carcinogens.

Serum-responsive expression of carbonyl-metabolizing enzymes in normal and transformed human buccal keratinocytes.

Gene expression of carbonyl-metabolizing enzymes (CMEs) was investigated in normal buccal keratinocytes (NBK) and the transformed buccal keratinocyte lines SVpgC2a and SqCC/Y1. Studies were performed at a serum concentration known to induce terminal squamous differentiation (TSD) in normal cells. Overall, 39 of 58 evaluated CMEs were found to be expressed at the transcript level. Together the transformed cell lines showed altered transcription of eight CME genes compared to NBK, substantiating earlier results. Serum increased transcript levels of ALDH1A3, DHRS3, HPGD and AKR1A1, and decreased those of ALDH4A1 in NBK; of these, the transformed, TSD-deficient cell lines partly retained regulation of ALDH1A3 and DHRS3. Activity measurements in crude cell lysates, including relevant enzymatic inhibitors, indicated significant capacity for CME-mediated xenobiotic metabolism among the cell lines, notably with an increase in serum-differentiated NBK. The results constitute the first evidence for differential CME gene expression and activity in non-differentiated and differentiated states of epithelial cells.

Insights into possibilities for grouping and read-across for nanomaterials in EU chemicals legislation.

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

Repair of DNA damage caused by formaldehyde in human cells.

The alkaline elution technique was used to study repair of DNA damage caused by formaldehyde (HCHO) in human bronchial epithelial cells and fibroblasts, skin fibroblasts, and DNA excision repair-deficient skin fibroblasts from donors with xeroderma pigmentosum. Exposure of cells to HCHO resulted in DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB) in all cell types. DPC were induced at similar levels and were also removed by all cell types, with a half removal time of 2 to 3 hr. HCHO caused more SSB in the normal cell types than in the xeroderma pigmentosum fibroblasts. However, in all cell types, including the xeroderma pigmentosum cells, HCHO-induced DNA SSB and DPC were removed at comparable rates. By excision repair of HCHO-induced DNA damage, normal cells generated SSB that were also readily repaired. HCHO was only moderately cytotoxic to normal bronchial epithelial cells and fibroblasts at concentrations that induced substantial DNA damage. HCHO enhanced the cytotoxicity of both ionizing radiation and N-methyl-N-nitrosourea in both cell types. The results indicate that most DPC caused by HCHO can be removed without the involvement of DNA excision repair. Furthermore, HCHO also directly causes DNA SSB as well as SSB generated indirectly during ultraviolet-type excision repair. These studies indicate the complexity of the HCHO-induced DNA damage and its repair and that HCHO may enhance the cytotoxicity of chemical and physical carcinogens in human cells.

A cyclin D1/cyclin-dependent kinase 4 binding site within the C domain of the retinoblastoma protein.

Phosphorylation of the retinoblastoma protein (Rb) by the cyclin D1/cyclin-dependent kinase (cdk) 4 complex (cdk4/D1) is a key regulatory step for maintaining the orderly progression of the cell cycle. The B domain of Rb contains a site that recognizes and binds the LXCXE motif found in D-type cyclins. This interaction is important for phosphorylation of Rb by cdk4/D1, although in vitro the Rb C domain alone is efficiently phosphorylated by cdk4/D1. A mutation in the C domain of Rb, L901Q, has been identified that completely abolishes cdk4/D1 phosphorylation of the isolated C domain. By contrast, the L901Q mutation has no effect on phosphorylation by either cyclin E/cdk2 or cyclin B/cdk1, suggesting that the interaction between L901Q and cdk4/D1 is specific. Introduction of the L901Q mutation into Rb containing the A, B, and C domains results in phosphorylation becoming predominantly dependent on the LXCXE binding region. However, when the LXCXE binding region of Rb is mutated, phosphorylation becomes dependent on the L901 site within the C domain. The L901 binding site can supplant the LXCXE binding site for the cdk4/D1-dependent phosphorylation of S780 and S795 but not S807/S811. Despite the limited homology between C domains of Rb, p107, and p130, the L901 site is conserved and introduction of the L925Q mutation into the isolated C domain of p107 also inhibits phosphorylation by cdk4/D1. These data support a model for cdk4/D1 recognizing two independent binding sites in Rb and suggests a conservation of this C domain binding motif for cyclin D1/cdk4 kinase among the Rb family of proteins.

O6-alkylguanine-DNA alkyltransferase activity in normal human tissues and cells.

Normal adult human tissues and cultured bronchial epithelial cells and fibroblasts exhibit O6-alkylguanine-DNA alkyltransferase activity in vitro by catalyzing the repair of the promutagenic alkylation lesion O6-methylguanine from DNA. The amount repaired by extracts of liver, peripheral lung, and colon extracts was proportional to the amount of extract protein. Repair of O6-methylguanine led to stoichiometric regeneration of guanine in the DNA and stoichiometric formation of S-methylcysteine in protein. Alkyltransferase activity varies in the different human tissues tested in the decreasing order of liver greater than colon greater than esophagus greater than peripheral lung greater than brain. Extracts of lung tissues, cultured human bronchial epithelial cells, and fibroblasts had similar alkyltransferase activities. Various human tissues exhibit 2- to 10-fold higher alkyltransferase activity than corresponding rat tissues. Whereas the interindividual variation of the activity was 4- to 5-fold in ten or more human lung and colon specimens, the interindividual variation in the inbred rat was less than 20%. The present results show that different human tissues and cells have a several-fold higher capacity to repair O6-methylguanine in DNA than do rat tissues and that the repair process occurs via a mechanism similar to that shown previously in other mammalian cells and Escherichia coli.

Effects of formaldehyde, acetaldehyde, benzoyl peroxide, and hydrogen peroxide on cultured normal human bronchial epithelial cells.

The effects of several aldehydes and peroxides on growth and differentiation of normal human bronchial epithelial cells were studied. Cells were exposed to formaldehyde, acetaldehyde, benzoyl peroxide (BPO), or hydrogen peroxide (HPO). The effect of each agent on the following parameters was measured: (a) clonal growth rate; (b) squamous differentiation; (c) DNA damage; (d) ornithine decarboxylase activity; (e) nucleic acid synthesis; (f) aryl hydrocarbon hydroxylase activity; and (g) arachidonic acid and choline release. None of the agents were mitogenic, and their effects were assessed at concentrations which reduced growth rate (population doublings per day) to 50% of control. The 50% of control concentrations for the 6-h exposure were found to be 0.065 mM BPO, 0.21 mM formaldehyde, 1.2 mM HPO, and 30 mM acetaldehyde. BPO-exposed cells were smaller than controls (median cell planar area, 620 sq microns versus 1150 sq microns), and acetaldehyde-exposed cells were larger than controls (median cell planar area, 3200 sq microns). All agents increased the formation of cross-linked envelopes and depressed RNA synthesis more than DNA synthesis. HPO caused DNA single-strand breaks, while formaldehyde and BPO caused detectable amounts of both single-strand breaks and DNA-protein cross-links. Other effects included increased arachidonic acid and choline release due to HPO. The similarities and differences of the effects of these aldehydes and peroxides to those caused by tumor promoters are discussed.

Rat and human explant metabolism, binding studies, and DNA adduct analysis of benzo(a)pyrene and its 6-nitro derivative.

Human colon and bronchus tissue explants were incubated with either [3H]benzo(a)pyrene ([3H]BP) or [3H]-6-nitrobenzo(a)pyrene ([3H]-6-NBP). The total percentage of metabolism of BP and 6-NBP was, respectively, 8-59% and 18-41% in bronchus and 11-23% and 36-50% in colon. A product tentatively identified as 3-hydroxy-6-NBP was isolated from the 6-NBP incubation medium. BP and 6-NBP when incubated at equivalent concentrations were found to bind covalently to the DNA of human bronchi from 15 cases at means of 42 and 50.9 pmol/10 mg DNA, respectively, and to the DNA of human colon from 6 cases at means of 66.5 and 35 pmol/10 mg DNA, respectively. The range among individuals was within one order of magnitude. High pressure liquid chromatography (HPLC) of enzymic hydrolysates of human bronchus explant DNA revealed one adduct from the BP-incubated bronchus which cochromatographed with (+/-)-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene-deoxyguanosine and a possible two adducts from the 6-NBP-incubated bronchus which eluted earlier than did the BP adduct. DNA obtained from the lung or liver of rats given 2.0-mg/kg doses of either [3H]BP or [3H]-6-NBP by i.p. injection was also enzymically hydrolyzed and analyzed on HPLC. Three DNA adducts were observed in liver and two were observed in lung DNA hydrolysates from rats given injections of [3H]BP. One adduct from each organ cochromatographed with (+/-)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene- deoxyguanosine; however, the major adduct in each case eluted earlier. Only one adduct was detected in liver and lung DNA hydrolysates from rats given [3H]-6-NBP, and this had the same retention time as did the major adduct isolated from human bronchus that had been incubated previously with [3H]-6-NBP. Salmonella typhimurium TA98 was incubated with [3H]-6-NBP and Aroclor-induced rat liver S9. Enzymically hydrolyzed DNA analyzed by HPLC revealed three adducts, two of which cochromatographed with the two DNA adducts isolated from human bronchus DNA adduct which had the same retention time as did the major liver and lung DNA adduct from rats given i.p. injections of [3H]-6-NBP. In each case the major adduct from DNA hydrolysates of rat liver and lung, human bronchus, and S. typhimurium, all treated with [3H]-6-NBP, cochromatographed with the major DNA adduct isolated from liver and lung DNA of rats given [3H]BP.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of benzo(a)pyrene and 1-naphthol in cultured human tumorous and nontumorous colon.

The oxidative metabolism of benzo(a)pyrene and the conjugative metabolism of 1-naphthol by explant cultures of normal human colon and colonic tumor tissue, obtained at surgery, have been studied. After 24 hr in culture, the explants were exposed to either [1-14C]-1-naphthol (20 to 100 microM) or [3H]-benzo(a) pyrene (1.5 microM) for a further 1.5 to 24 hr. Both normal-appearing tissue and tumor tissue metabolized benzo(a)pyrene to a wide variety of organic solvent-soluble metabolites, including monohydroxybenzo(a)pyrenes, dihydrodiols, and tetrols. 1-Naphthol was metabolized by cultured human colonic mucosa and tumor tissue to both its glucuronic acid and sulfate ester conjugates. In the normal tissues, with naphthol (20 microM), sulfate ester conjugation predominated. However, with the tumor tissue, sulfate ester conjugation decreased; thus, the percentage of glucuronic acid conjugates, expressed as a percentage of total metabolites formed, was increased significantly compared to normal tissue. The relationship, if any, of these changes to neoplastic transformation is unclear. The technique of explant culture described in this study may be of use for the study of other facets of the pathobiology of solid tumors.

Role of oxygen radicals in 12-O-tetradecanoylphorbol-13-acetate-induced squamous differentiation of cultured normal human bronchial epithelial cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits growth and induces terminal squamous differentiation of normal human bronchial cells when added to the culture media [J. C. Willey, A. J. Saladino, C. Ozanne, J. F. Lechner, and C. C. Harris, Carcinogenesis (lond.), 5: 209-215, 1984]. We have investigated the possibility of oxygen free radicals being involved as intermediates in this process. Electron paramagnetic resonance measurements using the spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide failed to detect oxygen free radicals in bronchial epithelial cells exposed to TPA, although oxy radicals were detected in bronchial epithelial cells after a nontoxic exposure to menadione, and in human neutrophils after exposure to TPA. Addition to the culture media of free radical scavenger, i.e., reduced glutathione, N-acetylcysteine, D-alpha-tocopherol, copper (II) (3,5-diisopropylsalicyclic acid)2, or the combination of superoxide dismutase and catalase did not affect the dose-dependent growth inhibition of TPA on the bronchial epithelial cells. Moreover, exposure of the bronchial epithelial cells to TPA did not result in increased DNA single strand breaks measured by alkaline elution, as would be expected with a free radical mediated mechanism. Thus, our results argue against the importance of oxygen free radicals in the inhibition of growth and the induction of squamous differentiation by TPA in normal human bronchial epithelial cells.

Biochemical and morphological effects of cigarette smoke condensate and its fractions on normal human bronchial epithelial cells in vitro.

We investigated the effect of cigarette smoke condensate (CSC), two basic fractions (BIa and BIb) of CSC, the ethanol-extracted weakly acidic fraction (WAe), and the methanol-extracted neutral fraction (Nmeoh) on the clonal growth rate, plasminogen activator (PA) activity, cross-linked envelope (CLE) formation, and ornithine decarboxylase activity, epidermal growth factor (EGF) binding, thiol levels, and DNA single strand breaks in cultured human bronchial cells. Neither CSC nor any of the fractions were mitogenic over the range 0.01-100 micrograms/ml. All were growth inhibitory at higher concentrations. The 40% growth inhibitory concentrations for CSC, BIa, BIb, WAe, and Nmeoh were 10, 10, 10, 3, and 1 micrograms/ml, respectively. Effects on CLE formation, morphology, PA, and ornithine decarboxylase activities, EGF binding, and thiol levels were evaluated using 40% growth inhibitory concentrations. We found that CSC and all fractions caused an increased formation of CLEs, from a baseline of 0.5% in the untreated cells to a maximum increase of 25% induced by Nmeoh. A squamous morphological change was observed within 1 h after exposure to Nmeoh, WAe, and CSC. The BIa and BIb fractions had little effect. Only Nmeoh increased PA significantly, from 2.5 +/- 0.4 to 5.1 +/- 0.3 units/mg cellular protein. CSC and the WAe and Nmeoh (Nmeoh greater than WAe greater than CSC) fractions caused a decrease in EGF binding, in each case reaching a maximum effect after a 10-12-h incubation. This effect on EGF binding was further characterized in the case of Nmeoh. In untreated normal human bronchial epithelial cells, by Scatchard analysis the kd was 2.0 nM and there were 1.2 X 10(5) receptors/cell. In cells incubated in medium containing Nmeoh (3 micrograms/ml) the kd was 3.2 nM and there were 1.1 X 10(5) receptors/cell. Thus, inhibition of EGF binding by Nmeoh was due primarily to a decrease in the affinity. At the 40% growth inhibitory concentrations neither CSC nor any of the fractions significantly affected intracellular thiol levels. While a 3-h incubation in medium containing CSC caused significant DNA single strand breaks only at a concentration of 100 micrograms/ml, Nmeoh caused a marked effect at 5 micrograms/ml. Neither CSC nor any of the fractions had an effect on ornithine decarboxylase activity. Due to the effects of the Nmeoh fraction on growth, morphology, EGF binding, PA activity, and formation of single strand breaks we consider it to be the most likely portion of CSC to contain compounds with actions similar to those of the phorbol ester, indole alkaloid, and polyacetate tumor promoters.

Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells.

Because betel quid chewing has been linked to the development of oral cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (arecoline, guvacoline, guvacine, and arecaidine), and four nitrosated derivatives [N-nitrosoguvacoline, N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionaldehyde and 3-(N-nitrosomethylamino)propionitrile] have been investigated using cultured human buccal epithelial cells. Areca nut extract in a dose-dependent manner decreases cell survival, vital dye accumulation, and membrane integrity, and it causes formation of both DNA single strand breaks and DNA protein cross-links. Depletion of cellular free low-molecular-weight thiols also occurs, albeit at quite toxic concentrations. Comparisons of the areca nut-related N-nitroso compounds and their precursor alkaloids, at concentrations up to 5 mM, indicate that 3-(N-nitrosomethylamino)propionaldehyde is the most potent on a molar basis to decrease both survival and thiol content and to cause significant formation of DNA single strand breaks. Arecoline, guvacoline, or N-nitrosoguvacoline decreases survival and cellular thiols, whereas arecaidine, guvacine, N-nitrosoguvacine, and 3-(N-nitrosomethylamino)propionitrile have only minor effects on these variables. Taken together, the present studies indicate that aqueous extract and, in particular, one N-nitroso compound related to areca nut, i.e., 3-(N-nitrosomethylamino)propionaldehyde, are highly cytotoxic and genotoxic to cultured human buccal epithelial cells, of potential importance in the induction of tumors in betel quid chewers.

Comparison of benzo(a)pyrene metabolism in bronchus, esophagus, colon, and duodenum from the same individual.

The metabolism of benzo(a)pyrene has been investigated in cultured normal human bronchus, colon, duodenum, and esophagus obtained from the same patient. The highest total metabolism was found in bronchus and duodenum, while the highest mean binding level was observed in the bronchus followed, in order, by the esophagus, duodenum, and transverse colon. A 30-fold interindividual variation in the binding level was found in each of the four organs studied, and a positive correlation between the binding levels in bronchus, colon, and duodenum was found. In human bronchus, a positive correlation was found between level of binding of benzo(a)pyrene to DNA and the amount of both benzo(a)pyrene 7,8-diol and the combined group of 3-hydroxybenzo(a)pyrene, benzo(a)pyrene 9,10-diol, and water-soluble metabolites. A significantly higher relative amount of benzo(a)pyrene tetrols and benzo(a)pyrene 9,10-diol was formed by human bronchus compared to the gastrointestinal tissues, while a higher level of benzo(a)pyrene phenols was formed by the latter. The relative distribution of benzo(a)pyrene-DNA adducts was similar in all four organs, the major DNA adduct being formed by trans-addition of anti-7,8-dihydroxy-9,10-epoxide-7,8,9,10-tetrahydrobenzo(a)pyrene to the 2-amino group at guanine. These results indicate that the metabolism of benzo(a)pyrene by at least four different organs is qualitatively similar but that quantitative differences exist.

Reactivity of fecapentaene-12 toward thiols, DNA, and these constituents in human fibroblasts.

Micromolar concentrations of fecapentaene-12, a mutagen found in human feces, decrease survival measured as colony-forming efficiency and membrane integrity of cultured human fibroblasts. Fecapentaene-12 also decreases the content of cellular free low-molecular-weight thiols including glutathione. Fecapentaene-12 reacts directly with glutathione by causing both decreased levels of free thiol and some concomitant formation of oxidized glutathione, indicating that thiol depletion is a result of both alkylation and oxidative reactions. Exposure of cells to 2 or 5 microM fecapentaene-12 causes significant amounts of DNA-interstrand cross-links and DNA-single strand breaks, respectively, whereas exposure to a higher concentration of fecapentaene-12, i.e., 10 microM, also causes significant DNA-protein cross-links. Results from the reaction of fecapentaene-12 with isolated plasmid DNA parallel the cellular pattern of DNA damage; primarily interstrand cross-links and strand breaks occur also in plasmid DNA. Taken together, these studies show that fecapentaene-12 is a potent cytotoxic and genotoxic agent which can react with cellular thiols and cause several types of DNA damage.

Pathobiological effects of acrolein in cultured human bronchial epithelial cells.

The ability of the highly reactive aldehyde acrolein to affect growth, membrane integrity, differentiation, and thiol status and to cause DNA damage has been studied at serum- and thiol-free conditions using cultured human bronchial epithelial cells. Acrolein markedly decreases colony survival at 3 microM whereas about 10-fold higher concentrations are required to increase membrane permeability, measured as uptake of trypan blue dye. Acrolein at micromolar concentrations also causes epithelial cells to undergo squamous differentiation as indicated by decreased clonal growth rate, dose-dependent increased formation of cross-linked envelopes, and increased cell planar surface area. Acrolein causes a marked and dose-dependent cellular depletion of total and specific free low-molecular-weight thiols as well as protein thiols. Exposure to acrolein did not cause oxidation of glutathione indicating that thiol depletion occurred by direct conjugation of reduced glutathione to acrolein without concomitant generation of active oxygen species. Furthermore, acrolein is genotoxic and causes both DNA single strand breaks and DNA protein cross-links in human bronchial epithelial cells. The results indicate that acrolein causes several cytopathic effects that relate to multistage carcinogenesis in the human bronchial epithelium.

Cloning of mutH and identification of the gene product.

A specialized lambda transducing phage carrying the mutH gene and several deletion derivatives of this phage were characterized by restriction enzyme analysis. This analysis localized the mutH gene to a small region of bacterial DNA on the transducing phage and facilitated the subsequent cloning of this gene into the multicopy plasmid pBR322. The mutH gene is contained entirely on a 1.5-kb HindIII fragment as judged by the ability of plasmids carrying this fragment to complement mutH- alleles on the bacterial chromosome. Using recombinant plasmids containing the 1.5-kb HindIII fragment, we identified an Mr 25000 protein as the product of the mutH gene in an in vitro transcription-translation system as well as in maxicells. Various deletion derivatives of the mutH-containing plasmids that exhibit a Mut- phenotype also have lost the Mr 25000 protein.

AP sites and AP endonucleases.

The sensitivity of internal and terminal apurinic/apyrimidinic (AP) sites to alkaline hydrolysis and beta-elimination is described. The symmetric and asymmetric modes of endonucleolytic hydrolysis by specific AP endonucleases are compared and the discrimination between their non-enzymatic and catalytic mechanism is discussed.