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Biomedical subjects

R G Harvey

Publications and source records attributed to R G Harvey.

At least 19 recordsLinked to original sources
PubMed1992

Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo[a]pyrene diol epoxide.

The unwinding of supercoiled phi X174 RFI DNA induced by the tumorigenic (+) and non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) has been investigated by agarose slab-gel and ethidium titration tube gel electrophoresis. The differences in adduct conformations were verified by flow linear dichroism techniques. Both enantiomers cause a reversible unwinding by the formation of noncovalent intercalative complexes. The effects of covalently bound BPDE residues on the electrophoretic mobilities of the RF I DNA form in agarose gels were investigated in detail in the range of binding ratios rb approximately 0.0-0.06 (covalently bound BPDE residues/nucleotide). In this range of rb values, there is a striking difference in the mobilities of (+)-BPDE- and (-)-BPDE-adducted phi X174 DNA in agarose slab-gels, the covalently bound (+)-BPDE residues causing a significantly greater retardation than (-)-BPDE residues. Increasing the level of covalent adducts beyond rb approximately 0.06 in the case of the (+)-BPDE enantiomer, leads to further unwinding and a minimum in the mobilities (corresponding to comigration of the nicked form and the covalently closed relaxed modified form) at rb 0.10 +/- 0.01; at still higher rb values, rewinding of the modified DNA in the opposite sense is observed. From the minimum in the mobility, a mean unwinding angle (per BPDE residue) of theta = 12 +/- 1.5 degrees is determined, which is in good agreement the value of theta = 11 +/- 1.8 degrees obtained by the tube gel titration method. Using this latter method, values of theta = 6.8 +/- 1.7 degrees for (-)-BPDE-phi X174 adducts are observed. It is concluded that agarose slab gel techniques are not suitable for determining unwinding angles for (-)-BPDE-modified phi X174 DNA because the alterations in the tertiary structures for rb < 0.06 are too small to cause sufficiently large changes in the electrophoretic mobilities. The major trans (+)-BPDE-N2-guanosine covalent adduct is situated at external binding sites and the mechanisms of unwinding are therefore different from those relevant to noncovalent intercalative BPDE-DNA complexes or to classical intercalating drug molecules; a flexible hinge joint and a widening of the minor groove at the site of the lesion may account for the observed unwinding effects. The more heterogeneous (-)-BPDE-nucleoside adducts (involving cis and trans N2-guanosine, and adenosine adducts) are less effective in causing unwinding of supercoiled DNA for reasons which remain to be elucidated.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

Analysis of point mutations in murine c-Ha-ras of skin tumors initiated with dibenz[a,j]anthracene and derivatives.

This study was designed to evaluate the point mutations in the murine c-Ha-ras gene of skin papillomas induced by initiation with dibenz[a,j]anthracene (DB[a,j]A), its bay-region anti-diol epoxide ((+/-)anti-DB[a,j]A-DE), and a 7,14-dimethyl analogue (7,14-diMeDB[a,j]A). Recent studies (Nair RV, et al., Chem Res Toxicol 4:115-122, 1991) in our laboratory have revealed both deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from the anti- and syn-diol epoxides of DB[a,j]A in cultured mouse epidermal cells after exposure to this hydrocarbon. Using PCR amplification and direct sequencing, we found specific A182----T transversion mutations (eight of 10 tumors) in codon 61 of c-Ha-ras in papillomas induced by initiation with DB[a,j]A. Analysis of papillomas generated by initiation with the more biologically potent analogue 7,14-diMeDB[a,j]A revealed that five of five tumors exhibited A182----T transversions in codon 61. The nature of the changes in the two DB[a,j]A tumors not showing codon 61 mutations in Ha-ras is currently not known since these tumor DNAs also did not possess c-Ha-ras mutations at codons 12, 13, or 59. Interestingly, papillomas produced by initiation with (+/-)anti-DB[a,j]A-DE also possessed A182----T transversion mutations in codon 61 of c-Ha-ras (five of five tumors). These data suggest that dAdo adducts derived from both parent hydrocarbons may play an important role in their tumor-initiating activity and possibly implicate a specific diol epoxide-dAdo adduct in this process.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Contribution of dihydrodiol dehydrogenase to the metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in fortified rat liver subcellular fractions.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) purified to homogeneity from rat liver cytosol will catalyze the NAD(P)(+)-dependent oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-diol) to yield benzo[a]pyrene-7,8-dione (BPQ). To verify that BPQ is a metabolite of B[a]P-diol in rat liver, an S100 fraction was supplemented with NAD+ and NADP+, and the formation of BPQ was followed by reverse-phase HPLC. The identity of BPQ was established by co-chromatography with an authentic standard (under different solvent conditions) and by RP-HPLC using a diode-array detector which established that the metabolite shared spectral identity with BPQ. The formation of BPQ in the S100 fraction was blocked by either a competitive inhibitor (indomethacin) or a suicide substrate [1-(4-nitrophenyl)-propen-1-ol] for DD, indicating that BPQ was being formed by this enzyme. To assess the contribution of DD to the metabolism of [3H]B[a]P-diol, subcellular fractions obtained from uninduced rat liver were fortified with co-factors to optimize the activity of enzymes that would compete for this proximate carcinogen. Under these conditions, S100 fractions fortified with NAD+ and NADP+ metabolized 25% of the B[a]P-diol, producing 731 +/- 154 pmol of BPQ. In contrast, rat liver microsomes fortified with an NADPH generating system metabolize 75% of the B[a]P-diol producing 2614 +/- 379 pmoles of benzo[a]pyrene-tetrahydrotetrols. Rat liver homogenates (S10) fortified with either uridine diphosphoglucuronic acid or phosphoadenosine phosphosulfate produced 180 +/- 56 and 95 +/- 31 pmoles of conjugates respectively, which were recovered as B[a]P-diol after treatment of the aqueous phase with either beta-glucuronidase or aryl sulfatase. Of the metabolites analyzed BPQ was formed in the second largest amount. These studies show that in uninduced rat liver DD may play a significant role in the metabolism of B[a]P-diol. The metabolic fate of BPQ remains to be determined.

Alcohol Oxidoreductases↗
PubMed1991

Preferential mutagenesis at G.C base pairs by the anti 3,4-dihydrodiol 1,2-epoxide of 7-methylbenz[a]anthracene.

The racemic anti-dihydrodiol epoxide of 7-methylbenz[a]anthracene preferentially induced mutations at G.C base pairs in the pS189 shuttle vector. Mutations were not randomly distributed throughout the supF target gene, but were concentrated at five hotspots. The hotspots for this agent did not correspond exactly to those produced by any other dihydrodiol epoxide examined to date, indicating that dihydrodiol epoxide structure and reactivity play a major role in determining mutagenic hotspots.

Base Composition↗
PubMed1991

Novel coumarins as potential anticarcinogenic agents.

The potential anticarcinogenic properties of several novel coumarin derivatives whose structures are based on polycyclic aromatic hydrocarbons (PAHs) were examined in the multistage model of mouse skin tumorigenesis. The test compounds were evaluated for their affinity to bind competitively with rat cytosolic Ah-receptor in rat hepatic cytosol, their effects on mouse epidermal aryl hydrocarbon hydroxylase (AHH) after topical application, and for their effects on the levels of hydrocarbon-DNA adducts formed in vivo. All compounds showed good correlations between cytosolic Ah-receptor binding and their ability to induce epidermal AHH activity. Among the derivatives evaluated the coumarin (8-methyl-9H-10-oxabenzo[a]pyren-9-one) exhibited the highest affinity for the Ah-receptor and was also the most potent inducer of epidermal AHH activity. This compound also effectively inhibited the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) to epidermal DNA when given either 5 min or 24 h prior to application of [3H]DMBA. This novel coumarin derivative significantly inhibited skin tumor initiation by DMBA in SENCAR mice when given at a dose of 200 nmol, 5 min (69% inhibition) or 24 h (76% inhibition) prior to initiation. The results of these studies suggest that this class of compounds shows considerable promise for future development as potential inhibitors of PAH-mediated tumor initiation on mouse skin. Potential mechanism(s) for the anti-initiating action of these compounds are discussed.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Spectroscopic characteristics and site I/site II classification of cis and trans benzo[a]pyrene diolepoxide enantiomer-guanosine adducts in oligonucleotides and polynucleotides.

The highly tumorigenic isomer (+)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] and its non-tumorigenic enantiomer (-)-anti-BPDE are known to react predominantly with the exocyclic amino group (N2) of deoxyguanine in DNA and to form adducts of different conformations. The spectroscopic characteristics (UV absorbance, fluorescence and circular dichroism) of stereochemically defined (+)-trans, (-)-trans, (+)-cis and (-)-cis d(5'-CACATGBPDETACAC) adducts in the single-stranded form, or complexed with the complementary strand d(5'-GTGTACATGTG) in aqueous solution, were investigated. The spectroscopic characteristics of the double-stranded d(5'-CACATGBPDETACAC).d(5'-GTGTACATGTG) adducts can be interpreted in terms of two types of conformations. In site I-type conformations, there is an approximately 10 nm red shift in the absorption maxima, which is attributed to significant pyrenyl residue-base interactions; in site II-type adducts, the red shift is only approximately 2-3 nm, and the pyrene ring system is located at external, solvent-exposed binding sites. The spectroscopic characteristics of the BPDE-modified duplexes are of the site II type for the (+)- and (-)-trans, and of the site I type for the (+)- and (-)-cis adducts. In adducts derived from the binding of (+)-anti-BPDE to poly(dG-dC).(dG-dC) and poly(dG).(dC), the trans/cis BPDE-N2-dG adduct ratio is 6 +/- 1; in the case of (-)-anti-BPDE this ratio is only 0.4 +/- 0.1 and 0.6 +/- 0.15 in poly(dG-dC).(dG-dC) and poly(dG).(dC) respectively. The spectroscopic properties of these BPDE-modified polynucleotide adducts are consistent with those of the BPDE-modified oligonucleotide complexes; the cis adducts are correlated with site I adduct conformations, while the trans adducts are of the site II type. The correlations between adduct characteristics and biological activities of the two BPDE enantiomers are discussed.

Base Sequence↗
PubMed1990

Relative tumor initiating activity of benzo[a]fluoranthene, benzo[b]fluoranthene, naphtho[1,2-b]fluoranthene and naphtho[2,1-a]fluoranthene on mouse skin.

The tumor-initiating activities of benzo[a]fluoranthene (BaF), benzo[b]fluoranthene (BbF), naphtho[1,2-b]fluoranthene (NbF) and naphtho[2,1-a]fluoranthene (NaF) were evaluated on the skin of female CD-1 mice. Each of these polycyclic aromatic hydrocarbons was assayed at total initiation doses of 1.0 and 4.0 mumol/mouse. These hydrocarbons were applied in 10 subdoses administered every other day. Promotion commenced 10 days after the last initiator dose and consisted of thrice weekly application of 2.5 micrograms of tetradecanoylphorbol acetate for 20 weeks. BbF was the most potent tumor initiator inducing a 100% incidence of tumor-bearing mice with an average of 8.5 tumors per mouse at a total initiator dose of 1.0 mumol. NaF was slightly more active as a tumor initiator than either NbF or BaF. NaF induced a 90% incidence of tumor-bearing mice with an average of 5.9 tumors per mouse at a total initiator dose of 1.0 mumol. BaF and NbF at a total initiator dose of 4.0 mumol exhibited similar tumor-initiating activity with both inducing a 90% incidence of tumor-bearing mice with an average of 4.3 and 6.6 tumors per mouse, respectively. However, at a total initiator dose of 1.0 mumol, BaF and NbF induced a 95% and 65% incidence of tumor-bearing mice with an average of 3.3 and 2.5 tumors per mouse, respectively.

Animals↗
PubMed1990

Age-, tissue-, and Ah genotype-dependent differences in the binding of 3-methylcholanthrene and its metabolite(s) to mouse DNA.

The induction of transplacental carcinogenesis by 3-methylcholanthrene (MC) in mice is determined, in part, by the genotype at the Ah locus. The relationship of Ah genotype and MC-induced DNA adducts was tested by comparing the response of pregnant and fetal C57BL/6 mice (Ahb Ahb; responsive to the induction of MC metabolism) and DBA/2mice (Ahd Ahd; nonresponsive). On day 17 of gestation (day 1 = presence of vaginal plug), C57BL/6 mice were treated i.p. with 100 mg/kg MC and DBA/2 mice with 30 mg/kg. Mice were sacrificed 24 h later and the tissues were analyzed for the presence of DNA adducts using the P1 nuclease version of the 32P-postlabeling method. With a 3.3-fold difference in administered dose, the total adduct levels in fetal DNA were (a) similar in both strains with the exception of liver, for which C57BL/6 mice had more adducts; (b) higher in the lung than skin, liver, or thymus; and (c) only 1/4 to 1/14 of the adult levels. Maternal DBA/2DNA contained more adducts in the thoracic lymph nodes and liver but fewer in the placenta and lung, compared to maternal C57BL/6 DNA. More adducts were detected in lung DNA than liver DNA in C57BL/6 mice. In contrast, these levels were similar in DBA/2 mice. When the difference in dose administered was considered in conjunction with this, less MC bound to DNA of C57BL/6 than DBA/2 mice overall. To identify adducts, oxidized metabolites of MC, 1-hydroxy-, 2-hydroxy-, 9,10-dihydrodiol-, or 3-methoxymethyl-MC, were topically applied to the dorsal skin of both strains. All of these metabolites produced adducts. Approximately 14 different adduct spots were detected. The two most abundant adducts were produced by 1-hydroxy-, 2-hydroxy-, and 9,10-dihydrodiol-MC. One of these also contained a 3-hydroxymethyl group. Several adducts did not contain the 9,10-dihydroxy group. The adducts derived from 3-methoxymethyl-MC were consistently found in greater abundance in DNA from C57BL/6 tissues, compared with DBA/2. Thus, oxidation of the 3-methyl group may be enhanced by Ah-dependent induction of MC metabolism. Together, these results suggest that the individual and total adduct levels are influenced by the genotype at the Ah locus, the route of administration, and the metabolite(s) with tissue and age specificity.

Animals↗
PubMed1990

Comparative tumor initiating activities of cyclopentano and methyl derivatives of 5-methylchrysene and chrysene.

Previous studies showed that 5,6-dimethylchrysene (5,6-diMeC) and 5,7-diMec were significantly less tumorigenic than 5-methylchrysene (5-MeC). These results were unexpected based on the known mechanism of metabolic activation of 5-MeC and indicated the presence of critical steric requirements for tumorigenicity at the 6 and 7 positions of 5-MeC. In this study, the structure activity relationships were further extended by comparing the tumor-initiating activities on mouse skin of 5-MeC, 6,7-cyclopentano-5-MeC, 5,6-diMeC, 6,7-diMeC, 5,7-diMeC, chrysene and 6,7-cyclopentanochrysene. 5-MeC was the most tumorigenic compound, with activity significantly higher than all other compounds tested. Among the other compounds. Only 5,6-diMeC was significantly tumorigenic. The results demonstrate that substitution of methyl or methylene groups at the 6 or 7 positions of 5-MeC leads to a significant reduction of tumor initiating activity.

Animals↗
PubMed1990

A connective tissue defect in two rabbits similar to the Ehlers-Danlos syndrome.

A connective tissue disease resembling the human Ehlers-Danlos syndrome is reported in two sibling rabbits about four months old. The clinical signs included skin hyperextensibility and fragility and poor wound healing. There were ultrastructural abnormalities in the structure of the collagen fibrils and in their arrangement in bundles of fibres. Affected rabbits may be a useful laboratory animal model for collagen disorders in human beings.

Animals↗
PubMed1990

Splicing mutations in the CHO DHFR gene preferentially induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene.

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 microM yielded at 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10(-4), 10(3)-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A.T----T.A and G.C----T.A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.

Animals↗
PubMed1990

Mutational specificity of the anti 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene.

An SV40-based pS189 shuttle vector, which contained a supF target gene and was replicated in human cells (Ad293), was used to determine the mutational specificity of anti 5-methylchrysene 1,2-dihydrodiol 3,4-epoxide, the active metabolite of the environmentally prevalent carcinogen 5-methylchrysene. The frequency of supF mutants containing point mutations increased with dose to approximately 40 times the spontaneous frequency. The induced mutations were not randomly distributed but occurred preferentially at mutagenic hotspots, which were not all identical to those reported by others for benzo[a]pyrene dihydrodiol epoxide, a metabolite with similar chemistry.

Base Sequence↗
PubMed1990

Preparation and isolation of adducts in high yield derived from the binding of two benzo[a]pyrene-7,8-dihydroxy-9,10-oxide stereoisomers to the oligonucleotide d(ATATGTATA).

The reaction of the (+)- and (-)-enantiomers of BDPE trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) with the oligodeoxynucleotide d(ATATGTATA) in aqueous buffer solutions gives rise predominantly to trans and cis addition products at the exocyclic amino group of the single deoxyguanosine residue. The trans/cis ratios are 7:1 in the case of (+)-BPDE, and 2:1 in the case of (-)-BPDE, while the reaction yields correspond to 34 and 15% respectively, of modified strands. These relatively high reaction efficiencies, at least for this particular type of oligonucleotide sequence, offer the possibilities of synthesizing relatively large amounts of well-defined covalent BPDE-oligonucleotide adducts (with different sequences of nucleotides flanking the modified base) for detailed spectroscopic and biochemical studies.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

Influence of the alkyl substituent on mutagenicity and covalent DNA binding of bay region diol-epoxides of 7-methyl- and 7-ethylbenz(a)anthracene in Salmonella and V79 Chinese hamster cells.

The anti-isomers of the bay region diol-epoxides of the strong carcinogen 7-methylbenz(a)anthracene and of the weak carcinogen 7-ethylbenz(a)anthracene were investigated for mutagenicity in Salmonella typhimurium (reversion of the his - strains TA98 and TA100 to prototrophy) and V79 Chinese hamster cells (acquisition of resistance to 6-thioguanine and ouabain; formation of micronuclei). In addition, in the V79 cells, the levels of the DNA adducts formed were determined by 32P-postlabeling analysis. In terms of mutations per nmol compound administered, the methyl derivative was four to 10 times more potent, depending on the genetic endpoint, than its ethyl congener. However, when the results were expressed as mutations per adduct, the difference between the two diol-epoxides was small. Therefore, a higher level of DNA modification appears to be the major reason for the stronger mutagenicity of the methyl derivative. However, both diol-epoxides had similar half-lives (about 9 min) in physiological buffer, as determined from the decline in mutagenic activity after preincubation of the test compound. These results suggest that the effect of the 7-alkyl group on the extent of reaction with DNA is more a result of steric factors than of a change in the intrinsic chemical reactivity of the diol-epoxides.

Animals↗
PubMed1989

Linear dichroism properties and orientations of different ultraviolet transition moments of benzo[a]pyrene derivatives bound noncovalently and covalently to DNA.

Linear dichroism and absorption methods are used to study the orientations of transition moments of absorption bands of polycyclic aromatic epoxide derivatives which overlap with those of the DNA band in the 240-300 nm region. Both the short and long axes of the pyrene residues of 1-oxiranylpyrene (1-OP) and the (+) and (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) noncovalently bound to double-stranded native DNA are oriented approximately perpendicular to the axis of the DNA helix, consistent with intercalative modes of binding. The covalent binding of these three epoxide derivatives to DNA is accompanied by reorientations of both the short and long axes of the pyrene residues. Covalent adducts derived from the highly mutagenic (+)-anti-BPDE are characterized by tilts of the short axis within 35 degrees or less, and of the long axis by more than 60-80 degrees, with respect to the planes of the DNA bases. In the adducts derived from the binding of the less mutagenic (-)-anti-BPDE and 1-OP epoxide derivatives to DNA, the long axes of the pyrenyl rings are predominantly oriented within 25 degrees of the planes of the DNA bases; however, in the case of the (-) enantiomer of BPDE, there is significant heterogeneity of conformations. In the case of the 1-OP covalent DNA adducts, the short axis of the pyrene ring system is tilted away from the planes of the DNA bases, and the pyrene ring system is not intercalated between DNA base-pairs as in the noncovalent complexes. The stereochemical properties of the saturated 7,8,9,10-ring in BPDE, or the lack of the 7 and 8 carbon atoms in 1-OP, do not seem to affect noncovalent intercalative complex formation which, most likely, is influenced mainly by the flat pyrenyl residues. These structural features, however, strongly influence the conformations of the covalent adducts, which in turn may be responsible for the differences in the mutagenic activities of these molecules.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗