Effects of consecutive thiouracil exposures in the juvenile and adult single comb White Leghorn chicken on body weight and reproductive performance

To develop a novel potent radical-scavenging antioxidant, the ideal structure of a phenolic compound was designed considering the factors that determine antioxidant potency. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4, 6-di-tert-butylbenzofuran (BO-653) was thus synthesized and its antioxidant activity was evaluated against lipid peroxidations in vitro. The electron spin resonance study showed that the phenoxyl radical derived from BO-653 was more stable than alpha-tocopheroxyl radical. BO-653 reduced alpha-tocopheroxyl radical rapidly, but alpha-tocopherol did not reduce the phenoxyl radical derived from BO-653. However, the chemical reactivity of BO-653 toward peroxyl radical was smaller than that of alpha-tocopherol. This was interpreted as the steric effect of bulky tert-butyl groups at both ortho positions which hindered the access of peroxyl radical to the phenolic hydrogen. However, the tertbutyl substituents increased the stability of BO-653 radical and also lipophilicity, and its antioxidant potency against lipid peroxidation in phosphatidylcholine liposomal membranes was superior to that of alpha-tocopherol. Ascorbic acid reduced the phenoxyl radical derived from BO-653 and spared BO-653 during the oxidation of lipid in the homogeneous solution. On the other hand, ascorbic acid did not spare BO-653 in the oxidation of liposomal membranes. It was concluded that BO-653 is a potent novel radical-scavenging antioxidant.     

Expression of topoisomerase II, bcl-2, and p53 in three human brain tumor cell lines and their possible relationship to intrinsic resistance to etoposide

We characterized three human brain tumor cell lines (D54, HBT-20, and HBT-28) with respect to resistance to etoposide (VP-16), a topoisomerase II-reactive drug. All three cell lines were inherently resistant to VP-16 when compared to other human cell lines, with D54 showing the greatest resistance using colony formation assays. Resistance to VP-16 has been attributed to decreased drug uptake and changes in topoisomerase II; however, drug uptake and topoisomerase II protein levels (immunoblot) were no lower in D54 than in HBT-20 and HBT-28, cell lines relatively more sensitive to VP-16. More to the point, measurement of topoisomerase II-mediated DNA cleavage of cellular DNA after treatment with VP-16 showed that the topoisomerase II in these cells was active. These data indicate mechanisms other than those attributable to decreased drug uptake or altered topoisomerase II exist for clinical resistance to VP-16. VP-16-induced DNA cleavage has been associated with apoptosis in some cell lines; however, neither DNA laddering nor morphological changes characteristic of apoptosis were detected in these cell lines after treatment with VP-16. Bcl-2 and mutant p53 were present in these cells. Either of these conditions can prevent apoptosis and could explain a dissociation between the proximal mediator of VP-16-induced cytotoxicity (topoisomerase II-DNA complex formation) and cellular death.     

Combination chemotherapy studies with gemcitabine and etoposide in non-small cell lung and ovarian cancer cell lines

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and etoposide (4'-demethylepipodo-phyllo-toxin-9-4,6-O-ethylidene-beta-D-g lucopyranoside, VP-16) are antineoplastic agents with clinical activity against various types of solid tumors. Because of the low toxicity profile of dFdC and the differences in mechanisms of cytotoxicity, combinations of both drugs were studied in vitro. For this purpose, we used the human ovarian cancer cell line A2780, its cis-diammine-dichloroplatinum-resistant and VP-16 cross-resistant variant ADDP, and two non-small cell lung cancer cell lines, Lewis Lung (LL, murine) and H322 (human). The interaction between the drugs was determined with the multiple drug effect analysis (fixed molar ratio) and with a variable drug ratio. In the LL cell line, the combination of dFdC and VP-16 at a constant molar ratio (dFdC:VP-16 = 1:4 or 1:0.125 after 4- or 24-hr exposure, respectively) was synergistic (combination index [CI], calculated at 50% growth inhibition = 0.7 and 0.8, respectively; CI <1 indicating synergism). After 24- and 72-hr exposure to both drugs at a constant ratio, additivity was found in the A2780, ADDP, and H322 cell lines (dFdC:VP-16 = 1:500 for both exposure times in these cell lines). When cells were exposed to a combination of dFdC and VP-16 for 24 or 72 hr, with VP-16 at its IC25 and dFdC in a concentration range, additivity was found in both the LL and H322 cells; synergism was observed in the A2780 and ADDP cells, which are the least sensitive to VP-16. Schedule dependency was found in the LL cell line; when cells were exposed to dFdC 4 hr prior to VP-16 (constant molar ratio, total exposure 24 hr), synergism was found (CI = 0.5), whereas additivity was found when cells were exposed to VP-16 prior to dFdC (CI = 1.6). The mechanism of interaction between the drugs was studied in more detail in the LL cell line; dFdCTP accumulation was 1.2-fold enhanced by co-incubation with VP-16, and was even more pronounced (1.4-fold) when cells were exposed to VP-16 prior to dFdC. dCTP levels were decreased by VP-16 alone as well as by the combination of both compounds, which may favor phosphorylation of dFdC, thereby increasing dFdCTP accumulation. DNA strand break (DSB) formation was increased for exposure to both compounds together compared to exposure to each compound separately, this effect being most pronounced when cells were exposed to VP-16 prior to dFdC (38% and 0% DSB for dFdC and VP-16 alone, respectively and 97% DSB for the combination). The potentiation in DSB formation might be a result of the inhibition of DNA repair by dFdC. Provided the right schedule is used, VP-16 is certainly a compound eligible for combination with dFdC.     

The use of transgenic animals in biotechnology

Conformational effects and affinities of VP-16 (etoposide) and its derivatives to DNA in the presence of Cu(II) ion were examined by circular dichroic (CD) spectra. The Cu(II)/Cu(I) redox kinetics and the hydroxyl radical (.OH) generation from the Cu(II)-complexes were estimated by the stopped-flow kinetics. Based on the results, DNA-cleaving activity of Cu(II)-complexes of VP-16 has been shown to be related with binding affinity of the complex to DNA, Cu(II)/Cu(I) redox and .OH generation, emphasising the mechanism of generated .OH attack to DNA.     

Effects of alkyl amine carboxyboranes on L1210 DNA fragmentation and nucleic acid metabolism

Amine-carboxyboranes with varying alkyl chain lengths were observed to be potent cytotoxic agents inhibiting the growth of a number of histological types of murine, rat, and human tumors. These agents preferentially reduced L1210 DNA synthesis with marked inhibition of the activities of regulatory enzymes of the purine pathway. Other enzyme activities which were marginally reduced were DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, t-RNA polymerase, and nucleoside kinases. Pyrimidine nucleotide pools were not reduced but DNA strand scission occurred after 24 h incubation with the agents. The amine-carboxyboranes were not DNA topoisomerase II inhibitors at 100 microM. The agents did not cause DNA protein linked breaks themselves; nevertheless, VP-16 [etoposide] induced DNA protein linked breaks were increased two fold in the presence of the agents suggesting synergistic effects. The amine-carboxyboranes decreased protein kinase C mediated phosphorylation of L1210 topoisomerase II protein, potentially decreasing its enzymatic catalytic activity. Thus, the amine-carboxyboranes did not function like VP-16 in affording cleavable products but were synergistic with VP-16 in causing DNA fragmentation. The agents were also additive with VP-16 in reducing tumor cell number, soft-agar colony growth and DNA synthesis and in producing DNA strand scission.     

Thalamic collaterals of corticostriatal axons: their termination field and synaptic targets in cats

We investigated the modification of etoposide (i.e. VP-16)-induced cell killing by hyperthermia in a radioresistant human melanoma (Sk-Mel-3) and a human normal (AG1522) cell line. VP-16, a DNA topo II poison, was given as a 1 h exposure at variable doses up to 35 microM; hyperthermia was given either before or following VP-16 treatment. Hyperthermic treatment comprised one of the following: 41 degrees C for 8 h, 42 degrees C for 2 h or 45 degrees C for 15 min. Hyperthermia preceding VP-16 treatment reduced the cytotoxicity of the latter; the reduction of VP-16 cytotoxicity was directly proportional to the severity of the hyperthermic treatment. For a particular combination of hyperthermic dose and VP-16 concentration, generally similar responses were seen in both cell lines. There were no effects on VP-16 cytotoxicity when both Sk-Mel-3 and AG1522 cells were heated at 41 degrees C for 8 h following treatment with VP-16. However, heating both cell lines at 45 degrees C for 15 min following VP-16 treatment again reduced the amount of cytotoxicity associated with VP-16. In addition, we found that a preceding exposure to 45 degrees C, 15 min heating did not affect either cellular accumulation or efflux of [3H]VP-16 in both cell lines. This suggested that the reduction in VP-16 cytotoxicity observed under those conditions was not due to a modification of VP-16 transport. We found no differences between the catalytic activities of topo II extracted from nuclei of Sk-Mel-3 and AG1522 cells that were either heated at 45 degrees C for 15 min or that were not subjected to such treatment. These results therefore suggested that the substantial reduction of cytotoxicity seen when 45 degrees C, 15 min heating preceded VP-16 treatment was also not due to an effect on topo II catalytic activity. Our results therefore demonstrate that hyperthermia, given either before or after VP-16, can actually reduce the amount of VP-16 cytotoxicity and that this can occur without any overt changes in VP-16 accumulation and efflux or in topo II catalytic activity.     

Effect of VP-16-213 on the intracellular degradation of DNA in HeLa cells

VP-16-213, a semisynthetic derivative of podophyllotoxin, is an active antitumor agent. In this paper, the effects of VP-16-213 and podophyllotoxin on microtubule assembly in vitro and nucleoside transport in HeLa cells are compared. At 100 muM, VP-16-213 does not inhibit microtubule assembly in vitro, while 5 muM podophyllotoxin completely prevents the formation of microtubules. The presence of the glucoside moiety in VP-16-213 is responsible for the inactivity of VP-16-213 in this system because 4'-demethylepipodo-phyllotoxin, the nonglucoside congener of VP-16-213, inhibits microtubule assembly. In HeLa cells, VP-16-213 and podophyllotoxin share a common biological property; both agents inhibit the uptake of thymidine and uridine into cells by inhibiting the facilitated diffusional component of nucleoside transport. The conncentrations of drug necessary to inhibit thymidine and uridine uptake into HeLa cells by 50% are 10 and 5 muM, respectively, for podophyllotoxin, and 25 and 20 muM for VP-16-213. The action of podophyllotoxin on nucleoside transport appears unrelated to its effect on microtubule assembly, since VP-16-213, which does not inhibit microtubule assembly, inhibits nucleoside transport.     

Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide

The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe; and (ii) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction.     

Evidence that a GABAA-like receptor is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa

Endogenous alpha-tocopherol of low density lipoprotein (LDL) particles exposed to ferrylmyoglobin (iron in the form of FeIV = O) vanishes as a function of myoglobin concentration. After alpha-tocopherol depletion, subsequent heavy lipid peroxidation is prevented by caffeic and p-coumaric acids, i.e., phenolic acids present in foods and beverages, by a mechanism involving the one-electron transfer reaction between the phenols and the ferrylmyoglobin, with formation of metmyoglobin and the corresponding phenoxyl radicals from caffeic and p-coumaric acids, as previously discussed. Caffeic acid delays alpha-tocopherol consumption when present before oxidation challenging and restores alpha-tocopherol when added halfway during the reaction. Conversely, p-coumaric acid accelerates the rate of alpha-tocopherol consumption when added either before or during the oxidation reaction. In LDL enriched with alpha-tocopherol, caffeic acid induces an inhibition period of oxidation longer than that expected from the sum of discrete periods characteristic of the phenolic acid and alpha-tocopherol. Surprisingly, p-coumaric acid decreases the peroxidation chain rate. Similar effects of these phenolic acids on alpha-tocopherol consumption were observed in a Triton X-100 micellar system, i.e., in the absence of a peroxidation chain reaction. Results suggest that caffeic acid acts synergistically with alpha-tocopherol, extending the antioxidant capacity of LDL by recycling alpha-tocopherol from the alpha-tocopherol radical (i.e., alpha-tocopheroxyl radical). By contrast, the phenoxyl radical from p-coumaric acid (produced by electron-transfer reaction between phenolic acid and ferrylmyoglobin) oxidizes alpha-tocopherol. However, in spite of alpha-tocopherol consumption, the exchange reaction recycling p-coumaric acid can still afford an antioxidant protection to LDL on basis of the chain-breaking activity of p-coumaric acid. These results emphasize the biological relevance of small structural modifications of phenols on the interaction with alpha-tocopherol in LDL. The significance of these results in the context of atherosclerosis is discussed.     

Rapid polymerase chain reaction assay to detect variation in the extent of gene-specific damage between cisplatin- or VP-16-resistant and sensitive lung cancer cell lines

We previously established a rapid and facile polymerase chain reaction (PCR)-stop assay for quantitation of specific gene damage in very small numbers of cells. The present study investigated whether the PCR-stop assay was able to detect variation in the extent of DNA damage in transcribed active genes between cisplatin- or VP-16-resistant and sensitive cells. The assay demonstrated that about twice as much genetic damage occurs in PC-9 cells than in cisplatin-resistant PC-9/CDDP cells following cisplatin exposure and about 4.6 times more damage occurs in H69 than in VP-16-resistant H69/VP cells following VP-16 exposure. These results show that DNA damage, as detected by PCR-stop assay, correlates with cytotoxicity. In conclusion, the PCR-stop assay could be useful in detecting variation in DNA damage in specific genes.     

Long-term trends in tuberculosis. Comparison of age-cohort data between Hong Kong and England and Wales

Apoptosis is cellular suicide functionally opposite of mitosis. It plays an important role in tissue growth control and removal of damaged and premalignant cells. The decrease in death suppressor Bcl-2 protein level was implicated in the many types of apoptotic cell death. Because Bcl-2 protein was recently found to be cleaved during apoptosis induced by Fas ligation, IL-3 withdrawal, and alphavirus infection, we assessed whether Bcl-2 protein was also cleaved during the anticancer drug (VP-16)-induced apoptotic cell death in U937 cells. We found that Bcl-2 protein was cleaved in vivo and in vitro after the treatment of VP-16. We also found that caspase-3/CPP32, which was activated after VP-16 treatment, was responsible for the direct cleavage of Bcl-2 protein. The overexpression of the cleaved Bcl-2 fragment increased the sensitivity to VP-16 and promoted apoptotic cell death. Therefore, caspase-3/CPP32 accelerates VP-16-induced U937 cell apoptosis by cleaving death suppressor Bcl-2 protein to produce a death promoter Bcl-2 fragment.     

The responses of secondary endings of cat soleus muscle spindles to succinyl choline

In this report we examine biochemical and genetic alterations in DNA topoisomerase II (topoisomerase II) in K562 cells selected for resistance in the presence of etoposide (VP-16). Previously, we have demonstrated that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreased stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoisomerase II/DNA complex formation, and contains reduced mRNA and protein levels of the M(r) 170,000 isoform of topoisomerase II, compared with parental K562 cells. K/VP.5 cells grown in the absence of VP-16 for 2 years maintained resistance to VP-16, decreased levels of topoisomerase II, and attenuated ATP stimulation of VP-16-induced topoisomerase II/DNA binding, compared with K562 cells. Sequencing of cDNA coding for two consensus ATP binding sites and the active site tyrosine in the K/VP.5 topoisomerase II gene indicated that no mutations were present in these domains. In addition, single-strand conformational polymorphism analysis of restriction fragments encompassing the entire topoisomerase II cDNA revealed no evidence of mutations in the gene for this enzyme in K/VP.5 cells. Nuclear extracts from K562 (but not K/VP.5) cells contained a heat-labile factor that potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in isolated nuclei from K/VP.5 cells. Immunoprecipitated topoisomerase II from K/VP.5 cells was 2.5-fold less phosphorylated, compared with enzyme from K562 cells. Collectively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regulates topoisomerase II phosphorylation and hence drug-induced topoisomerase II/DNA covalent complex formation and stability.     

A regulatory role for recombinase activating genes, RAG-1 and RAG-2, in T cell development

Effects of etoposide (VP-16) and cytosine arabinoside (Ara-C) on the cell cycle of HL-60 and THP-1 cells were studied by flow cytometry using the bromodeoxyuridine (BrdU)/DNA assay technique to investigate the efficacy of VP-16 for monocytic leukemia cells. VP-16 inhibited the proliferation of THP-1 cells more strongly than that of HL-60 cells at any concentrations used at 24 and 48 hr. VP-16 arrested HL-60 and THP-1 cells in the G2/M phase and reduced them in the G0/G1 and early S phase at higher concentrations. There was no significant difference in the percentage of G2/M phase cells at the same concentration between both cells. However, reduction in the G0/G1 and early S phase cells was more marked in THP-1 than HL-60 cells significantly. On the other hand, Ara-C perturbed the cell cycle of HL-60 cells more than that of THP-1 cells at 24 and 48 hr. These results suggest that the effects of VP-16 on the cell cycle may be more intense in THP-1 than HL-60 cells, and support the efficacy of VP-16 for treating monocytic leukemia in vivo.     

Decreased drug accumulation without increased drug efflux in a novel MRP-overexpressing multidrug-resistant cell line

KB/7D cells represent a multidrug-resistant subclone of human nasopharyngeal carcinoma KB cells generated by continuous exposure to the topoisomerase II inhibitor VP-16 (etoposide). KB/7D cells also show cross-resistance to doxorubicin and vincristine. Phenotypic traits of the cell line include a 2-fold decrease in topoisomerase II levels and a decrease in the uptake of VP-16 without an increase in the rate of drug efflux or expression of P-glycoprotein, suggesting a novel mechanism associated with the uptake of anticancer drugs. This study demonstrated that the multidrug-resistance associated protein (MRP) is overexpressed in KB/7D cells, and that the loss of resistance in revertant cells correlates with the loss of MRP. The resistance to VP-16 and doxorubicin could be overcome, partially, and resistance to vincristine could be overcome completely, by the L-enantiomer of verapamil, but not by the D-enantiomer or by BIBW 22 (4-[N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-2,7-bis[cis-2,6-++ +dimethylmorpholino)-6-phenylpteridin), an inhibitor of MDR-1. L-Verapamil was shown to be significantly more potent than D-verapamil in modulating the accumulation defect in KB/7D cells towards doxorubicin, as measured by flow cytometry and confocal microscopy, and towards VP-16, as measured by increases in protein-linked DNA strand breaks. This suggests that KB/7D cells are multidrug resistant due to decreases in topoisomerase II levels and the overexpression of MRP, that MRP leads to a decrease in drug accumulation, and that L-verapamil can modulate the MRP-associated accumulation defect and drug-resistance phenotype. This contrasts with previous studies that suggest that MRP causes multidrug resistance by exporting cytotoxic drugs out of the cell and that did not show modulation of MRP by verapamil.     

The effect of the chemotherapeutic drug VP-16 on poly(ADP-ribosylation) in apoptotic HeLa cells

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters; in particular we have focused on changes in cellular morphology that are considered as markers of apoptosis. By immunofluorescence experiments we have shown that VP-16 causes the complete disruption of nucleoli and induces chromatin margination and fragmentation. Agarose gel electrophoresis of DNA from cells treated with 10-100 microM VP-16 showed the appearance of a characteristic ladder due to the internucleosomal DNA cleavage. The effect of etoposide on DNA integrity was not prevented by preincubation of cells with the protein synthesis inhibitor cycloheximide. These results provide experimental evidence indicating that the typical features of apoptosis are visible in HeLa cells exposed to VP-16. In this experimental system we have investigated whether the ADP-ribosylation process could be regulated by the presence of DNA fragments. By means of the activity gel technique, which allows the direct evaluation of automodified poly(ADP-ribose)polymerase, we have observed that in extracts from cells where etoposide-induced DNA fragmentation occurred, the autoribosylated form of the enzyme is greatly increased. Ribosylated poly(ADP-ribose)polymerase has been isolated by affinity chromatography on boronate column from cells permeabilized and labelled with [32P]NAD. Drug exposure caused a strong augmentation of modified enzyme. These observations suggest that activation of ADP-ribosylation process occurs in cells that show the typical features of apoptosis.     

Unusual substituent effects in the hydroxylation of phenols by a Cu(2+)-ascorbic acid-O2 system, gamma-radiolysis, and microsomes

Aromatic ring hydroxylation of phenolic compounds proceeded selectively in a Cu(2+)-ascorbic acid-O2 system. In contrast to usual oxidation systems, unusual substituent effects were observed: electron-withdrawing groups accelerate and electron-donating groups retard the hydroxylation. Hydroxyl radicals generated by gamma-radiolysis also converted phenols into catechols and hydroquinones. The close resemblance of these two systems led us to presume that the active intermediate in the Cu(2+)-ascorbic acid-O2 system is hydroxyl radical or an equivalent species. The unusual substituent effects were also observed in the hydroxylation of p-substituted phenol by microsomes, a cytochrome P-450-dependent reaction. These results imply that the Cu(2+)-ascorbic acid-O2 system is an effective biomimetic oxidation system.     

Peroxidase-mediated oxidation, a possible pathway for activation of the fungal nephrotoxin orellanine and related compounds. ESR and spin-trapping studies

Orellanine is the tetrahydroxylated and di-N-oxidized bipyridine toxin extracted from several Cortinarius mushrooms among them C. orellanus. The pathogenic mechanism involved in the C. orellanus-poisoning by orellanine leading to kidney impairment is not yet fully understood until now. Electron spin resonance (ESR) spectroscopy has been used to study the activation of orellanine by horseradish peroxidase/H2O2 system at physiological pH. Evidence for a one-electron oxidation of the toxin by this enzymatic system to an ortho-semiquinone radical intermediate is presented. The orellanine ortho-semiquinone generated by the peroxidase/H2O2 system abstracts hydrogen from glutathione, generating the glutathionyl radical which is spin-trapped by 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and subsequently detected by ESR spectroscopy. Similarly, the ortho-semiquinone abstracts hydrogen from ascorbic acid to generate the ascorbyl radical which is detected by direct ESR. The peroxidatic oxidation of orellanine to semiquinone followed by its reduction by glutathione or ascorbic acid does not induce dioxygen uptake. The relationship between chemical structure and HRP oxidation of orellanine-related molecules, namely orelline and DHBPO2 (the parent molecule lacking of hydroxyl groups in 3 and 3' position) has been investigated in absence or in presence of reducing agents. None of the orellanine-related compounds can be oxidized by the HRP/H2O2 system, showing that both catecholic moieties and aminoxide groups are necessary for observing the formation of the ortho-semiquinone form of orellanine. As shown for the (photo)chemical oxidation of orellanine, the mechanism of toxicity could be correlated with a depletion of glutathione and ascorbate levels which are implicated in the defence against oxidative damage.     

Identification of the site of the globin-derived radical in leghaemoglobins

Reaction of the Fe3+ form of the oxygen-carrying protein leghaemoglobin (MetLb), derived from the root nodules of lupins, with H2O2 is shown to generate, in addition to an iron (IV)-oxo (ferryl) species, a globin radical. This radical has been detected by EPR spectroscopy and is analogous to the species previously observed with the soybean protein. Analysis of the hyperfine coupling constants and g value of the EPR signal, together with computer simulations and the similarity of the observed spectra of that detected with the soybean form suggest that this species is also a tyrosine-derived phenoxyl radical; this species is believed to arise via an electron-transfer process within the protein with an electron being transferred from the tyrosine residue to an initially-generated Compound-1-type species. Comparison of the protein sequences and structures of the two proteins show that there is only one conserved tyrosine residue (at position 133 in the soybean and 138 in the lupin); this is believed to be the site of the phenoxyl radical. The lupin phenoxyl radical reacts with added water-soluble antioxidants and reducing agents which result in repair of the radical; this may be an important protective mechanism in vivo. Analysis of molecular models of the protein structures is in accord with both the assignment of the radical to this conserved tyrosine residue and the observed radical reactivity.     

ESR studies of a series of phthalocyanines. Mechanism of phototoxicity. Comparative quantitation of O2-. using ESR spin-trapping and cytochrome c reduction techniques

ESR experiments with 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP) and the spin-trap 5,5-dimethyl pyrroline-N-oxide (DMPO) have been performed on a series of new phthalocyanines: the bis(tri-n-hexylsiloxy) silicon phthalocyanine ([(nhex)3SiO]2SiPc), the hexadecachloro zinc phthalocyanine (ZnPcCl16), the hexadecachloro aluminum phthalocyanine (AlPcCl16), the hexadecachloro aluminum phthalocyanine sulfate (HSO4AlPcCl16), whose photocytotoxicity has been studied against various leukemic and melanotic cell lines. Type I and Type II pathways occur simultaneously in DMF although the Type II seems to be prevalent. These results are not changed when the bis(tri-n-hexylsiloxy) silicon phthalocyanine is entrapped into liposomes. By contrast, the Type I process is favored in membrane models for all the perchlorinated phthalocyanines. This modified behavior may be accounted on a possible stacking of phthalocyanines in membranes and a preventing effect of axial ligands against aggregation in the case of the bis(tri-n-hexylsiloxy) silicon phthalocyanine. The photodynamic action of zinc perchlorinated phthalocyanine is not dependent on singlet oxygen, phototoxicity of this molecule being essentially mediated by oxygen free radicals. Quantitation of the superoxide radical was accomplished, with good agreement, by two techniques: the cytochrome c reduction and the ESR quantitation based on the double integration of the first derivative of the ESR signal. The disproportionation of the superoxide radical or degradation of the spin-trap seem to be avoided in aprotic solvents such as DMF.     

Effect of inhibitors of poly(ADP-ribose) polymerase on the induction of GRP78 and subsequent development of resistance to etoposide

We have recently demonstrated that cell lines deficient in poly(ADP-ribose) synthesis due to deficiency in the enzyme poly(ADP-ribose) polymerase (PADPRP) or depletion of its substrate NAD+ overexpress GRP78. Furthermore, this overexpression of GRP78 is associated with the acquisition of resistance to topoisomerase II-directed drugs such as etoposide (VP-16); (S. Chatterjee et al., Cancer Res., 54: 4405-4411, 1994). Thus, our studies suggest that interference with NAD+-PADPRP metabolism could provide an important approach to (a) define pathways of GRP78 induction, (b) study the effect of GRP78 on other cellular processes, (c) elucidate the mechanism of GRP78-dependent resistance to topoisomerase II targeted drugs, and (d) modulate responses to chemotherapy in normal and tumor tissues. However, in the in vivo situation, it is impractical to interfere with NAD+-PADPRP metabolism by mutational inactivation of PADPRP or by depletion of its substrate NAD+. Therefore, we have examined several inhibitors of NAD+-PADPRP metabolism including 3-aminobenzamide, PD128763, and 6-aminonicotinamide for their ability to reproduce the results obtained with cell lines deficient in NAD+-PADPRP metabolism relative to the induction of GRP78 and subsequent development of resistance to VP-16. Our studies show that 6-aminoicotinamide treatment is highly effective in the induction of GRP78 and subsequent development of resistance to VP-16, whereas treatment with 3-aminobenzamide or PD128763 does not induce GRP78 and thus does not result in VP-16 resistance.