Roger A. Lewis

Genetic toxicology of bleomycin.

Bleomycin (BLM), an antibiotic obtained from Streptomyces verticillus, is of significance as an antineoplastic agent. The compound is actually the mixture of some 200 related forms which differ from each other in the amine moiety. The drug, at low concentrations, can cause elimination of bases, particularly thymine. This causes strand breakage of DNA and inhibition of cell growth. The influence of BLM on cell growth may be unrelated to the effects on DNA. In general, mitotically dividing cells show more DNA damage than non-dividing cells. G2 seems to be the most sensitive phase indicating that cell death may not be related to a direct effect of BLM on DNA replication. The antibiotic shows specific effects on chromatin and causes chromosomal damage in all sub-phases of interphase. It can affect early prophase chromosomes also. Suggestion has been made that BLM-induced breakage and cell death are similar to those induced by densely ionizing radiations. Whereas the antibiotic affects the frequency of somatic crossing over and produces micronuclei, the data on mutation induction and production of sister-chromatid exchanges do not permit classifying BLM as a potent inducer of these phenomena. The genetic effects of BLM can be modified quantitatively by thiol compounds, caffeine, hyperthermia and H2O2. It is concluded that the available data do not permit assessment of genetic damage in the offsprings of BLM-treated patients. Such studies are urgently needed, as are the studies to find out the effects of BLM on meiotic phenomena.

Phosphorylation of arabinosyl guanine by a mitochondrial enzyme of bovine liver

Extracts of mitochondria isolated from bovine liver were shown to phosphorylate araG, forming araGMP as the sole product. When other nucleosides were used as competitors with araG as the substrate for phosphorylation, deoxycytidine, deoxythymidine and guanosine were not significantly inhibitory. However, the phosphorylation of araG was blocked by deoxyguanosine, deoxyadenosine and deoxyinosine. Deoxyguanosine was shown to be a competitive inhibitor of araG phosphorylation (apparent Ki for deoxyguanosine = 9 microM; apparent Km for araG = 66 microM). Likewise, araG was determined to be a competitive inhibitor of mitochondrial deoxyguanosine kinase activity (apparent Km for deoxyguanosine = 16 microM; apparent Ki for araG = 55 microM). These data suggest that the two nucleosides were phosphorylated by the same enzyme. Disc gel electrophoresis showed that the phosphorylating activity for araG migrated with deoxyguanosine kinase activity. The pH profiles of the araG and deoxyguanosine kinase activities were dissimilar. The optimum pH for deoxyguanosine kinase was 5.5; for araG kinase, it was 8.0. Collectively, these data suggest that araG is phosphorylated by mitochondrial deoxyguanosine kinase; however, separate forms of the enzyme or different reaction conditions may be involved in the optimal activities of the two catalytic events.

Inhibition of DNA polymerase by captan

Captan inhibits DNA polymerases of both eukaryotic and prokaryotic sources. When polymerases were employed in assays with various nucleotides as template-primer, no specificity in the base sequence of polynucleotide was required for inhibition. Sucrose gradient centrifugation and preincubation studies showed the inhibition was caused by an irreversible alteration of the polymerase. Captan and DNA compete for the same site on the polymerase, thus DNA can serve a protective role in the elimination of captans action. The pyrophosphate exchange activity associated with the polymerase is not inhibited by captan and the fidelity with which DNa polymerase I copies the DNA template also is not altered by captan treatment.

The metabolism of deoxyguanosine in mitochondria. Characterization of the uptake process

SummaryThe uptake of deoxyguanosine by rat liver mitochondria was characterized. The process required an intact mitochondrial membrane and exhibited a dependence on added phosphate. Deoxyguanosine uptake was minimally influenced by Mg2+ or Mn2+, but Ca2+ at concentrations above 0.5 mM were detrimental. Of the deoxynucleosides tested, only deoxyinosine inhibited the uptake of deoxyguanosine. The ribonucleoside guanosine was not observed to compete with its deoxynucleoside analog. Known inhibitors of nucleoside transport, cytochalasin B and NBMPR, did not block deoxyguanosine uptake, but the sulfhydryl reagents NEM and pCMB were both inhibitory. The uptake of deoxyguanosine was shown to be a saturable process and an apparent Km of 0.64 σM was calculated from a Hanes plot.

Inhibition of DNA polymerase β activity in isolated bovine liver nuclei by captan and related compounds

Abstract The effects on DNA synthesis of the fungicide captan and several structurally related compounds were investigated in isolated bovine liver nuclei. Captan, folpet, captafol, and trichloromethanesulfenyl chloride inhibited DNA synthesis to the same degree with ID 50 values of approximately 50 μ M in a 40-min assay. The inhibition is concentration dependent and the degree of inhibition increases with time. Studies with structural analogs of captan indicated that inhibition of DNA synthesis by captan is mediated through the trichloromethylthio moiety of the captan molecule. In addition, the data indicate thiophosgene is probably not the toxic species involved in the inhibition of DNA synthesis. The isolated nuclei used in this study were shown to exhibit only a single DNA polymerase activity which was determined to be of the β or low-molecular-weight type. In addition to its inhibition in intact nuclei, captan inhibited the activity of the β polymerase in nuclear extracts as well as in partially purified enzyme preparations. These results indicate that captan inhibits DNA synthesis in our preparation of isolated nuclei by acting directly on the DNA polymerase-catalyzed reaction rather than by causing a nonspecific or indirect effect in the nuclear system such as alterations in the nuclear membrane or aggregation of the nuclei. The site of captans inhibitory action is the DNA polymerase molecule. The interaction of captan with the polymerase results in irreversible inhibition of the enzyme. Interaction of captan with the template, if it occurs, does not appear to be involved in mediating the inhibition.

Interaction between activated nordihydroguaiaretic acid and deoxyribonucleic acid.

Abstract Nordihydroguaiaretic acid (NDGA) is a natural product of the lignan family that has been shown to possess antimicrobial and antineoplastic properties in a variety of test systems. NDGA was observed by u.v.-visible spectroscopy to be unstable in an aqueous environment; however, by these same techniques NDGA was shown to be stable in the presence of mercaptoethanol. It is suggested that an activated NDGA is an intermediate in the O 2 -mediated oxidation of NDGA and that the activated NDGA forms a stable complex when reacted with duplex DNA. This DNA-activated NDGA complex was detected and studied by both fluorescence spectroscopy and CsCl density gradient techniques. The addition of DNA quenched the fluorescence of activated NDGA in a concentration-dependent fashion. Furthermore, the exposure of activated NDGA lowered the buoyant density of DNA, also, in a concentration-dependent manner. Since mononucleotides did not quench the fluorescence of activated NDGA, and heat-denatured DNA was less effective than fully duplex DNA in its ability to interact with activated NDGA, the duplex structure of DNA was determined to be important in the complex formation. Whereas activated NDGA bound to both poly dG · poly dC and poly (dA · dT), there appeared, by one method of analysis, to be a preference for poly dG · poly dC. Activated NDGA-DNA complex was stable to dialysis, but dissociated in the presence of Sarkosyl. No changes in the viscosity or melting temperature of DNA was induced by the addition of activated NDGA. These data suggest a mechanism in which the activated NDGA was bound to the more apolar regions of duplex DNA that are located in either the major and/or minor grooves.

Phosphorylation of deoxyguanosine in intact and fractured mitochondria.

The phosphorylation of deoxyguanosine was measured in fractured and intact mitochondria and an apparent Km of 16 μM for deoxyguanosine was calculated using fractured mitochondria. The effects of various deoxynucleotides on the phosphorylating activity in fractured organelles was tested at both a high and low ratio of NXP/ATP and at two pH values, 7.0 and 5.5. Exogenous dGTP, dGDP or dITP were inhibitory under all conditions tested. With a NXP/ATP ratio of 0.08 at pH 7.0, TTP, TDP, dADP, ADP, UTP and UDP were stimulatory, but at pH 5.5 only TTP elicited that response. When the NXP/ATP ratio was 10 at pH 5.5, TTP and UTP increased the activity more than 10-fold, whereas, at pH 7.0 TTP, TDP, dADP, ADP, UTP, UDP caused stimulation, but to a much lesser extent. When exogenous Mg2+, Mn2+ or Ca2+ were added to intact mitochondria, the rates of phosphorylation were lowered. In fractured mitochondria in the absence of exogenous ATP, little phosphorylation occurs, hence these metal ions caused little change. ATP-Mg, ATP-Mn and ATP-Ca, each at 0.05 mM caused a small inhibition with intact mitochondria, whereas, these compounds supported phosphorylation with fractured organelles. ATP-Mn (10 mM) or ATP-Ca (10 mM) stimulated phosphorylation in both intact and fractured mitochondria. Intact mitochondria synthesized dGMP, dGDP and dGTP when metal ion or ATP-Me concentrations were low (0.05 mM) or when Mg2+ concentration was high (10 mM). Additions of ATP-Ca, ATP-Mn, ATP-Mg, Mn2+ or Ca2+ at 10 mM cause the loss of dGDP and dGTP formation and, in most cases, an increase in the synthesis of dGMP. Fractured mitochondria make only dGMP and the levels of its synthesis are greater than that observed for intact mitochondria. These data suggest that intact mitochondria are required for the synthesis of dGTP and that its synthesis is regulated by mitochondria nucleotides.

The partial purification and characterization of purine nucleoside phosphorylase from mammalian mitochondria.

Cytosolic purine nucleoside phosphorylase (PNPase) is a well known, and described enzyme which exists in a variety of organisms, both procaryotic and eucaryotic. More recently this enzyme was found in bovine liver mitochondria. The mitochondrial purine nucleoside phosphorylase was purified 63 fold and has a molecular weight of 48–60 kD. From Lineweaver-Burk plots apparent Kms of 23μM for inosine, 42 μM for deoxyinosine, 40 μM for phosphate, 2 μM for hypoxanthine, and 163 μM for ribose-1-phosphate were calculated. Both 8-aminoguanosine (Ki=0.5 μM) and araG (Ki=381 μM) are inhibitors of the enzyme. The proteins isoelectric point (pI) was calculated at a pH of 4.2. Preliminary immunological work showed no cross-reactivity between epitopes on the mitochondrial protein and those on PNPase from human erythrocytes. The apparent Kms calculated for the mitochondrial enzyme are,with the exception of that using hypoxanthine, within the range commonly associated with Kms from the cytosolic species. The mitochondrial enzymes molecular weight and pI are less than normally described. The enzymes isolation from mitochondria, together with several unique characteristics, suggest that it is a separate protein from that found in the cytosol.

The metabolism of deoxyguanosine in mitochondria: a characterization of the phosphorylation process which occurs in intact mitochondria.

Isolated, intact mitochondria were evaluated for their ability to phosphorylate deoxyguanosine. This activity was stimulated by exogenous ATP, substrates for oxidative phosphorylation or added inorganic phosphate. Inhibitors of oxidative phosphorylation lowered the levels of deoxyguanosine phosphorylation. From a Hanes plot, an apparent Km of 0.83 microM deoxyguanosine was calculated for the phosphorylation activity in intact mitochondria. In the presence of a 20-fold excess of added deoxynucleosides, none of those tested were strongly inhibitory. However, added UDP and dTDP were stimulatory and dGTP and dGDP were inhibitory to the phosphorylation of deoxyguanosine. These data show that mitochondria phosphorylate deoxyguanosine and that the process is regulated by other events which take place within the organelle.

Enhancement of deoxyguanosine kinase activity in human lung fibroblast cells infected with human cytomegalovirus

SummaryStudies were conducted to establish the relationship between deoxyguanosine kinase activity and human cytomegalovirus (HCMV) infection. Using both PAGE and isoelectric focusing techniques, extracts from untreated and infected cells were examined for deoxyguanosine kinase activity. The analyses resulted in identical migration rates for deoxyguanosine kinase activity in both infected and uninfected extracts. These data and kinetic studies based on apparent Km values suggest that HCMV enhanced a cellular kinase activity rather than coded for a virus specific enzyme. Furthermore, our results indicated that infected cells, like normal fibroblasts, contain two deoxyguanosine kinase activities, one of mitochondrial and another of cytosolic origin. Of particular interest was the observation that HCMV infection caused an enhancement of the mitochondrial enzymatic activity while the cytosolic activity showed no change. Deoxycytidine kinase activity which is associated with cytosolic deoxyguanosine kinase was unaffected by HCMV infection.