Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[a]- pyrene diol epoxide adducts at the 6-amino group of adenine

DNA topoisomerase I (top1) is the target of potent anticancer agents, including camptothecins and DNA intercalators, which reversibly stabilize (trap) top1 catalytic intermediates (cleavage complexes). The aim of the present study was to define the structural relationship between the site(s) of covalently bound intercalating agents, whose solution conformations in DNA are known, and the site(s) of top1 cleavage. Two diastereomeric pairs of oligonucleotide 22-mers, derived from a sequence used to determine the crystal structure of top1–DNA complexes, were synthesized. One pair contained either a trans-opened 10R- or 10S-benzo[a]pyrene 7,8-diol 9,10-epoxide adduct at the N6-amino group of a central 2′-deoxyadenosine residue in the scissile strand, and the other pair contained the same two adducts in the nonscissile strand. These adducts were derived from the (+)-(7R,8S,9S,10R)- and (−)-(7S,8R,9R,10S)-7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. On the basis of analogy with known solution conformations of duplex oligonucleotides containing these adducts, we conclude that top1 cleavage complexes are trapped when the hydrocarbon adduct is intercalated between the base pairs flanking a preexisting top1 cleavage site, or between the base pairs immediately downstream (3′ relative to the scissile strand) from this site. We propose a model with the +1 base rotated out of the duplex, and in which the intercalated adduct prevents religation of the corresponding nucleotide at the 5′ end of the cleaved DNA. These results suggest mechanisms whereby intercalating agents interfere with the normal function of human top1.

Chronic, topical exposure to benzo[a]pyrene induces relatively high steady-state levels of DNA adducts in target tissues and alters kinetics of adduct loss.

Carcinogen-DNA adduct measurements may become useful biomarkers of effective dose and/or early effect. However, validation of this biomarker is required at several levels to ensure that human exposure and response are accurately reflected. Important in this regard is an understanding of the relative biomarker levels in target and nontarget organs and the response of the biomarker under the chronic, low-dose conditions to which humans are exposed. We studied the differences between single and chronic topical application of benzo[a]pyrene (BAP) on the accumulation and removal of BAP-DNA adducts in skin, lung, and liver. Animals were treated with BAP at 10, 25, or 50 nMol topically once or twice per week for as long as 15 weeks. Animals were sacrificed either at 24, 48, or 72 hr after the last dose at 1 and 30 treatments, and after 24 hr for all other treatment groups. Adduct levels increased with increasing dose, but the slope of the dose-response was different in each organ. At low doses, accumulation was linear in skin and lung, but at high doses the adduct levels in the lung increased dramatically at the same time when the levels in the skin reached apparent steady state. In the liver adduct, levels were lower than in target tissues and apparent steady-state adduct levels were reached rapidly, the maxima being independent of dose, suggesting that activating metabolism was saturated in this organ. Removal of adducts from skin, the target organ, was more rapid following single treatment than with chronic exposure. This finding is consistent with earlier data, indicating that some areas of the genome are more resistant to repair. Thus, repeated exposure and repair cycles would be more likely to cause an increase in the proportion of carcinogen-DNA adducts in repair-resistant areas of the genome. These findings indicate that single-dose experiments may underestimate the potential for carcinogenicity for compounds that follow this pattern.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato1

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.

Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development.

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.

Traumatic brain damage prevented by the non-N-methyl-D-aspartate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f] quinoxaline.

The mechanisms of neuronal degeneration following traumatic head injury are not well understood and no adequate treatment is currently available for the prevention of traumatic brain damage in humans. Traumatic head injury leads to primary (at impact) and secondary (distant) damage to the brain. Mechanical percussion of the rat cortex mimics primary damage seen after traumatic head injury in humans; no animal model mimicking the secondary damage following traumatic head injury has yet been established. Rats subjected to percussion trauma of the cortex showed primary damage in the cortex and secondary damage in the hippocampus. Morphometric analysis demonstrated that both cortical and hippocampal damage was mitigated by pretreatment with either the N-methyl-D-aspartate (NMDA) antagonist 3-((+/-)- 2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) or the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX). Neither treatment prevented primary damage in the cortex when therapy was started after trauma. Surprisingly, delayed treatment of rats with NBQX, but not with CPP, beginning between 1 and 7 hr after trauma prevented hippocampal damage. No protection was seen when therapy with NBQX was started 10 hr after trauma. These data indicate that both NMDA- and non-NMDA-dependent mechanisms contribute to the development of primary damage in the cortex, whereas non-NMDA mechanisms are involved in the evolution of secondary damage in the hippocampus in rats subjected to traumatic head injury. The wide therapeutic time-window documented for NBQX suggests that antagonism at non-NMDA receptors may offer a novel therapeutic approach for preventing deterioration of the brain after head injury.

Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

The octadecanoic pathway: signal molecules for the regulation of secondary pathways.

Plant defense against microbial pathogens and herbivores relies heavily on the induction of defense proteins and low molecular weight antibiotics. The signals between perception of the aggression, gene activation, and the subsequent biosynthesis of secondary compounds are assumed to be pentacylic oxylipin derivatives. The rapid, but transient, synthesis of cis-jasmonic acid was demonstrated after insect attack on a food plant and by microbial elicitor addition to plant suspension cultures. This effect is highly specific and not caused by a number of environmental stresses such as light, heavy metals, or cold or heat shock. Elicitation of Eschscholtzia cell cultures also led to a rapid alkalinization of the growth medium prior to jasmonate formation. Inhibition of this alkalinization process by the protein kinase inhibitor staurosporine also inhibited jasmonate formation. The induction of specific enzymes in the benzo[c]phenanthridine alkaloid pathway leading to the antimicrobial sanguinarine was induced to a qualitatively and quantitatively similar extent by fungal elicitor, methyl jasmonate, and its linolenic acid-derived precursor 12-oxophytodienoic acid. It is herein proposed that a second oxylipid cascade may exist in plants starting from linoleic acid via 15,16-dihydro-12-oxophytodienoic acid to 9,10-dihydrojasmonate. Experiments with synthetic trihomojasmonate demonstrated that beta-oxidation is not a prerequisite for biological activity and that 12-oxophytodienoic acid and derivatives are most likely fully active as signal transducers. Octadecanoic acid-derived compounds are essential elements in modulating the synthesis of antibiotic compounds and are thus integral to plant defense.