3 HBenzo a pyrene and seven isolated 3 Hbenzo a pyrene metabolites were incubated
with liver microsomes from control, phenobarbital-, and 3-methylcholanthrene-treated
rats, and irreversible binding ...to microsomal proteins was studied. For all compounds
tested, including benzo a pyrene-4,5-oxide, the binding was greatly enhanced in the
presence of a NADPH-generating system. All metabolites except 3-hydroxybenzo a -pyrene bound more extensively to microsomal proteins from 3-methyicholanthrene-treated rats than to proteins from phenobarbital-treated
or control rats. Benzo a pyrene-7,8-dihydrodiol bound much more efficiently than the other metabolites. The protein
binding patterns of the metabolites were examined by SDS-polyacrylamide gel electro-phoresis and fluorography. The most extensive
binding occurred to a few proteins in the
MW region of 45,000-70,000, and there were differences in the patterns between different
metabolites. The composite pattern of the metabolites corresponded to the binding
pattern obtained with benzo a pyrene. A major target protein in microsomes from 3-methylcholanthrene-treated rats had the same mobility as purified cytochrome
P -448.
Most binding to microsomes from phenobarbital-treated animals occurred to a 60,000
MW component. Binding also occurred to a protein which comigrated with cytochrome
P -450. The specificity and extent of metabolite binding to proteins may be of importance
for the development of cytotoxicity. Factors affecting the binding patterns are discussed.
A new procedure for obtaining rat lung microsomes essentially free of interfering hemoproteins has been developed. The method includes Sepharose 2B column chromatography of the 12,000 X g supernatant ...of lung homogenates, followed by ultracentrifugation of the material eluted in the void volume. Microsomes isolated in this manner contain specific levels of cytochromes b5 and P-450 and of NADPH-cytochrome c reductase that are among the highest ever reported for a rat lung microsomal fraction. After treatment of rats with 3-methylcholanthrene, the specific content of cytochrome P-450 in lung microsomes is doubled and that of cytochrome b5 increases 1.5 times. Several spectral differences between hepatic and lung microsomal cytochrome P-450 are apparent. In lung microsomes, the maximum of the reduced CO-bound cytochrome complex in a difference spectrum is at 453 nm for the noninduced hemoprotein and shifts to 451 nm after 3-methylcholanthrene induction. In contrast, no significant change in the ethylisocyanide difference spectra of reduced microsomes is obtained after induction; moreover, the spectra obtained with induced and noninduced cytochrome P-450 are similar to the one shown by hepatic microsomes from polycyclic hydrocarbon-treated rats. Furthermore, spectrophotometric studies on n-octylamine binding to control and induced lung cytochrome P-450 yielded results different from those previously obtained with rabbit liver microsomes. It is concluded that the cytochrome P-450 present in rat lung microsomes before and after 3-methylcholanthrene treatment of the animals is distinctly different from the liver hemoprotein.
The oligonucleotide 5'-d(CCTATAGATATCC) has been reacted with the (+)- or (-)-enantiomers of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzoapyrene (+)- and (-)-anti-BPDE respectively. ...Consistent with previous studies employing single-stranded oligonucleotides, adduct formation of both anti-BPDE enantiomers preferentially involved trans-addition of the C10 position of the diol-epoxide to the exocyclic nitrogen of deoxyguanosine in the following abbreviated as (+)-BPDEt-N2-G and (-)-BPDEt-N2-G adducts respectively. The unmodified or (+)-BPDEt-N2-G-modified oligonucleotide was allowed to form duplexes with the complementary sequence 5'-d(GGATATCTATAGG) or sequences in which C has been replaced with T, G or A and analysed with regard to thermal stability. The presence of a (+)-BPDEt-N2-G adduct in oligonucleotide duplexes substantially decreased the value of the melting point relative to the corresponding unmodified duplex. In mismatched complexes containing the (+)-BPDEt-N2-G adduct, a further decrease in thermal stability was observed. The presence of a (+)-BPDEt-N2-G adduct did not seem to change the extent of hyperchromicity (approximately 20%) upon melting. 5'-d(GGATATCTATAGG) or strands in which C was replaced with T, G or A were gradually added to (+)- or (-)-BPDEt-N2-G-modified oligonucleotides and the fluorescence emission intensity was determined. In all cases with (+)-BPDEt-N2-G, except when C was replaced with A in the complement, the fluorescence intensity steadily decreased and became constant at equal strand concentrations. When a strand containing A in place of C was gradually added to the (+)-BPDEt-N2-G oligonucleotide, a marked increase in the fluorescence intensity was observed (> 3-fold). In contrast, addition of strands containing A, T or G to the (-)-BPDEt-N2-G-modified oligonucleotide increased the fluorescence intensity from 1.5- to > 5-fold. Addition of the fully complementary sequence to the (-)-BPDEt-N2-G-containing oligonucleotide resulted in reduced fluorescence, however less pronounced than with the (+)-BPDEt-N2-G-modified analogue. Significant changes in spectral properties of the adducts were observed in the duplexes. The absorption and fluorescence excitation maxima of the single-stranded (+)-BPDEt-N2-G-modified oligonucleotide were at 353 nm. Insertion of C or A opposite the adduct caused a significant shift of these maxima to shorter wavelengths (347-348 nm). Addition of acrylamide, a fluorescence quencher, reduced the fluorescence intensity in all cases, but to variable extents.
Chromatin isolated from Ehrlich ascites cells was incubated with the tumourigenic compound (+)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzapyrene (+)-anti-BPDE at low ionic strength and the ...modified chromatin was analysed using flow linear dichroism (LD). The results confirm that (+)-anti-BPDE preferentially binds to the DNA in the linker regions, and furthermore show that the long axis of the bound pyrenyl chromophore is oriented parallel or close to parallel to the average orientation of the chromatin fiber axis. The data indicate that the binding geometry of (+)-anti-BPDE in chromatin is similar to that in pure DNA and deoxyguanosine-containing double-helical oligonucleotides.
The structure of the covalent (+)-anti-BPDE-poly(dG-dC) complex can be represented by two preferred orientations of the pyrene moiety; one at about 20 degrees relative to the helix axis and one at ...about 70 degrees, populated as 4:1. A rapid mobility of the BPDE may allow an exchange between the two orientations. The poly(dG-dC) structure becomes more flexible by (+)-anti-BPDE modification, seen as a shortened persistence length. This complex may be significant as a model for DNA interaction with covalently binding polyaromatic carcinogens.
Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzoapyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to ...excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.
Sodium nitrite was shown to enhance the metabolism of trans-7,8-dihydroxy-7,8-dihydrobenzoapyrene (BP-7,8-diol) to 7/8,9,10- and 7,10/8,9-tetrahydroxy-7,8,9,10-tetrahydrobenzoapyrene (tetraols) in ...phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMNs). The production of these tetraols implicates the intermediate formation of the corresponding trans-7,8-dihydroxy-9,10-epoxy-7,8-9,10-tetrahydrobenzoapyrene (anti-BPDE). A 2- to 3-fold increase in the tetraol yield was observed in the presence of nitrite in excess of 1 mM. Sodium azide, an inhibitor of myeloperoxidase and catalase, reduced the nitrite-stimulated metabolism of BP-7,8-diol in PMA-activated leukocytes. Diphenylene iodonium sulphate, a NADPH-oxidase inhibitor, lowered the production of tetraols in PMA-stimulated leukocytes both in the absence and presence of nitrite. Additionally, nitrite markedly enhanced the covalent binding of metabolites derived from 3H(-)-BP-7,8-diol to leukocyte proteins as well as to DNA present extracellularly. The nitrite-stimulated covalent binding to both proteins and DNA was inhibited by the presence of sodium azide. The mechanism underlying the effect of nitrite on the metabolism of BP-7,8-diol to reactive intermediates in PMA-activated human polymorphonuclear leukocytes is not known. However, the results are compatible with a peroxidase-dependent mechanism although other possible pathways may contribute to the enhanced rate of metabolism.
The transition from B to Z conformation has been studied in poly(dG-dC) covalently modified with racemic anti- or syn-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a strong and a weak ...carcinogen, respectively. Circular dichroism was used to study the kinetics of the transition after a sudden increase of the ionic strength to 2.7 M NaCl. The results show that the rate of the B to Z transition of poly(dG-dC) in high NaCl concentration is considerably enhanced by bound anti-BPDE and diminished by bound syn-BPDE. The results may be interpreted such that at the binding site of anti-BPDE the base stacking is distorted and made looser, which facilitates the B to Z transition. The partly intercalative nature of the syn-BPDE complexes apparently is effective in reducing the rate of the transition. These properties of the two BPDEs may be relevant to explain their different carcinogenic potencies.
![Interaction of benz[a]pyren...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0014-5793_1873-3468/small "Interaction of benz[a]pyrene diol epoxide with chromatin studied by flow linear dichroism")
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![Dynamics of benzo[a]pyrene ...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0301-4622/small "Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC).(dG-dC) studied by synchrotron excited fluorescence polarization anisotropy decay")
