Mechanisms of reaction of benzo(a)pyrene-7,8-diol-9,10-epoxide with DNA in aqueous solutions

The physical and chemical reaction pathways of the metabolite model compound benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) in aqueous (double-stranded) DNA solutions was investigated as a function of temperature (0-30 degrees C), pH (7.0-9.5), sodium chloride concentration (0-1.5M) and DNA concentration in order to clarify the relationships between the multiple reaction mechanisms of this diol epoxide in the presence of nucleic acids. The reaction pathways are (1) noncovalent intercalative complex formation with DNA, characterized by the equilibrium constant K, and Xb the fraction of molecules physically bound; (2) accelerated hydrolysis of BPDE bound to DNA; (3) covalent binding to DNA; and (4) hydrolysis of free BPDE(kh). The DNA-induced hydrolysis of BPDE to tetraols and the covalent binding to DNA are parallel pseudo-first-order reactions. Following the rapid (millisecond time scale) noncovalent complex formation between BPDE and DNA, a much slower (approximately minutes) H+-dependent (either specific or general acid catalysis) formation of a DNA-bound triol carbonium ion (rate constant k3) occurs. At pH 7.0 the activation energy of k3 is 8.7 +/- 0.9 kcal/mol, which is lower than the activation energy of hydrolysis of free BPDE in buffer solution (14.2 +/- 0.7 kcal/mol), and which thus partially accounts for the acceleration of hydrolysis of BPDE upon complexation with DNA. The formation of the triol carbonium ion is followed by a rapid reaction with either water to form tetraols (rate constant kT), or covalent binding to DNA (kc). The fraction of BPDE molecules which undergo covalent binding is fcov approximately equal to kc/(kc + kT) = 0.10 and is independent of the overall BPDE reaction rate constant k = kh(1 - Xb) + k3Xb if Xb----1.0, or is independent of Xb as long as k3Xb much greater than kh(1 - Xb). Thus, at Xb = 0.9, fcov is independent of pH (7.0-9.5) even though k exhibits a 70-fold variation in this pH range and k----kh above pH 9 (k3 = kh). Similarly, fcov is independent of temperature (0-30 degrees C), while k varies by a factor of approx. 3. In the range of 0-1.5 M NaCl, fcov decreases from 0.10 to 0.04. These variations are attributed to a combination of salt-induced variations in the factors k3, Xb and the ratio kc/kT.     

Inhibition of growth and decreased survival of B104 rat neuroblastoma cells after exposure to hyperbaric oxygen

Summary The toxic effects of hyperbaric oxygen (HBO) on growth and survival of B104 rat neuroblastoma cells were investigated. Cells in log phase growth were incubated at 37°C with 10 atm O₂ for 1 to 4 h. After exposure to HBO, cells were monitored for their subsequent growth and survival. Two hours of exposure caused a slowing of growth, which returned to normaly by the end of the 7th d of the postexposure period. Exposures to O₂ of 3 h or longer caused a complete cessation of growth for 4 d after the exposure and very litle or no recovery after this period. Increased hydrostatic pressure for 6 h using helium as the inert gas had no effect on growth. A colony formation assay was used to quantitative the degree of cell death induced by HBO. The resulting survival curve was of the exponential type with a broad shoulder between 0 to 2.5 h of exposure to 10 atm O₂. The curve fell off sharply at 2.5 h with an exponential decrease in survival when the exposure to HBO was extended to 4 h. At 2 h about 50% of cells were killed, but at 4 h only 2% survived the treatment. These results show that the depression of the growth rate by HBO is related to the number of cells that are killed by the exposure. This system provides a model in which the molecular and cellular effects of HBO can be investigated. This work was supported by the National Institutes of Health, Bethesda, MD, Grants AM-21423 and CA-14489, and by grants from the W. W. Smith Foundation and the Philadelphia Foundation. The studies presented here are part of a dissertation submitted by G. J. Gendimenico in partial fulfillment of the requirements for the degree of Ph.D. in pharmacology from the University of Pennsylvania.     

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.     

Efficient resolution of the dihydrodiol derivatives of benzo[a]pyrene by high-pressure liquid chromatography of the related (−)-dimenthoxyacetates

The diastereomeric (−)-dimenthoxyacetate derivatives of (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene were efficiently resolved by high-pressure liquid chromatography (HPLC) on silica gel. Treatment with methanolic ammonia under mild conditions removed the menthoxyacetate groups to furnish the optically pure (+) and (−) enantiomers of the trans-dihydrodiol. Epoxidation of each of the latter with m-chloroperbenzoic acid gave the corresponding (−) and (+) anti-diolepoxides. The speed and efficiency of resolution of these relatively sensitive compounds by HPLC demonstrates the potential utility of this technique for resolution of all types of carcinogen-derived arene dihydrodiols.     

Model reactions of the quinone metabolites of carcinogenic hydrocarbons with t-butylthiol

Reactions of benzo[a]pyrene 1,6-dione with t-butylthiolate affords two products of conjugate addition and subsequent spontaneous reoxidation, namely, 2-t-butylthio- and 2,4-di-t-butylthiobenzo[a]pyrene 1,6-dione. Analogous reaction of benzo[a]pyrene 3,6-dione furnished only 12-t-butylthiobenzo[a]pyrene 3,6-dione. Structural assignments are based on analysis of the high-resolution 270-MHz Fourier-transform proton nmr spectra. In both products the attachment of the entering nucleophile is on an aromatic ring remote from either of the carbonyl groups, the first examples of such detected. The biological significance of these results with relation to the potential reactions of these quinones, known to be major metabolites of the carcinogen benzo[a]pyrene, with glutathione, cysteine, and proteins in vivo is discussed.     

The molecular size and shape of liver glycogen.

The molecular-weight distribution of liver glycogen has been established from the analysis of sedimentation rates of fractions separated on sucrose density gradients and from the direct measurement of the diffusion coefficients of these fractions by laser-intensity-fluctuation spectroscopy. Hydrodynamic studies indicated that all fractions of glycogen of mol.wt.exceeding 25x10(6) had about 1.1 g of water per g of polysaccharide associated with them. The hydration and hydrodynamic behaviour of all fractions of mol.wt. exceeding 25x10(6) was similar, whereas smaller fractions behaved anomalously, indicating a substantially different overall structure.     

Yersinia Enterocolitica

An intragastric inoculation of approx. 2 × 1010 Yersinia enterocolitica cells killed chinchillas in three days in the case of four strains out of six tested. Because of the sensitivity of chinchillas to this bacterium, the test is useful for the evaluation of the virulence and invasiveness of Y. enterocolitica isolates. This animal model could also be used for studies on the mechanism of the infection.