A threshold concept for cancer therapy

Oxygen free radicals have been implicated in many disease processes, including aging and carcinogenesis, and have been associated with a variety of complications resulting from the treatment of cancer. As a result, the treatment of free radical-induced disease with antioxidants or free radical scavengers has become an important therapeutic modality. Ironically, these same oxygen free radicals also play a critical role in anti-cancer therapies. The use of antioxidants such as superoxide dismutase (SOD), in this setting, has been found to decrease the efficacy of anti-tumor therapies, which depend on free radical generation for their action. In addition, increased antioxidant activity can often be utilized by the tumor cell to favor increased growth. Therefore, the appropriate application of oxygen free radicals and antioxidants seems to be critically important in designing proper strategies for both prevention and treatment of malignant disorders. This review will summarize free radical and antioxidant regimens that have been employed to date, examine some of the problems associated with these regimens, introduce the 'threshold concept' explaining the dual effects of oxygen free radicals and antioxidants, and discuss a novel hypothesis regarding therapy that could potentially improve outcome in cancer patients.     

Use of catalytic topoisomerase II inhibitors to probe mechanisms of chemical-induced clastogenicity in Chinese hamster V79 cells

Determination of the clastogenic potential of new chemical entities, particularly pharmaceuticals, is an important part of the overall safety assessment of such drugs. It is appreciated that clastogenicity can arise from perturbation of many different cellular processes distinct from direct DNA/drug interactions. One such alternative clastogenic process is inhibition of DNA topoisomerase II, during which process the topoisomerase/DNA/drug ternary complex forms stable DNA double-strand breaks (cleavable complex), which become templates for recombinational, mutagenic, and chromosomal fragmentation events. Without extensive experimentation, it is generally not possible to distinguish clastogenicity arising from direct drug/DNA interaction from that arising from inhibition of topoisomerase II. In the present investigation, we demonstrate that specific catalytic inhibitors of DNA topoisomerase II reduce the clastogenicity of topoisomerase poisons but not that arising via non-topoisomerase-dependent mechanisms. In particular, it is shown that catalytic topoisomerase II inhibitors such as chloroquine, sodium azide, and A-74932, as well as certain intercalating agents such as 9-aminoacridine and ethidium bromide, strongly antagonize the formation of micronuclei induced by the DNA gyrase inhibitor clinafloxacin and the antitumor topoisomerase II poison etoposide. These catalytic inhibitors are also shown to antagonize the clastogenicity of experimental compounds and novel pharmaceuticals presumed to be DNA intercalating agents by virtue of their response in a cell-based bleomycin amplification assay. We extend our previous hypothesis, suggesting that the clastogenicity of some nonstructurally alerting drugs may be due to an as yet unappreciated propensity for DNA intercalation. It is further proposed that intercalation-dependent inhibition of DNA topoisomerase II may be responsible for this clastogenicity and that this may be detected in intact mammalian cells with the use of catalytic topoisomerase inhibitors.     

Anaerobic threshold: the concept and methods of measurement.

The anaerobic threshold (AnT) is defined as the highest sustained intensity of exercise for which measurement of oxygen uptake can account for the entire energy requirement. At the AnT, the rate at which lactate appears in the blood will be equal to the rate of its disappearance. Although inadequate oxygen delivery may facilitate lactic acid production, there is no evidence that lactic acid production above the AnT results from inadequate oxygen delivery. There are many reasons for trying to quantify this intensity of exercise, including assessment of cardiovascular or pulmonary health, evaluation of training programs, and categorization of the intensity of exercise as mild, moderate, or intense. Several tests have been developed to determine the intensity of exercise associated with AnT: maximal lactate steady state, lactate minimum test, lactate threshold, OBLA, individual anaerobic threshold, and ventilatory threshold. Each approach permits an estimate of the intensity of exercise associated with AnT, but also has consistent and predictable error depending on protocol and the criteria used to identify the appropriate intensity of exercise. These tests are valuable, but when used to predict AnT, the term that describes the approach taken should be used to refer to the intensity that has been identified, rather than to refer to this intensity as the AnT.     

In vitro evaluation of the clastogenicity of fumagillin

Fumagillin, an antibiotic compound produced by Aspergillus fumigatus, is effective against microsporidia and various Amoeba species, but is also toxic when administered systemically to mammals. Furthermore, a recent in vivo study by Stanimirovic Z et al. 2007: (Mutat Res 628:1-10) indicated genotoxic effects of fumagillin. The aim of the present study was to investigate and explain the clastogenic effects of fumagillin (in the form of fumagillin dicyclohexylamine salt) on human peripheral blood lymphocytes in vitro by sister-chromatid exchanges (SCE), chromosome aberrations (CA), and micronucleus (MN) tests. The mitotic index (MI), proliferation index (PI), and nuclear division index (NDI) were calculated to evaluate the cytotoxic potential of fumagillin. Five concentrations of fumagillin (0.34, 0.68, 1.02, 3.07, and 9.20 microg/ml) were applied to lymphocyte cultures. All the tested concentrations of fumagillin increased the frequency of SCE per cell significantly (P < 0.001 or P < 0.01) compared with the negative control. A significant (P < 0.001) increase in frequency of structural CA was observed at the three highest concentrations in comparison with the negative control. In addition, the three highest test concentrations increased MN formation and decreased MI, PI, and NDI significantly compared with the negative control. The present results indicate that fumagillin is clastogenic and cytotoxic to cultured human lymphocytes.     

Investigations into the fluctuations of proprioceptive reflexes in man

Summary During controlled, defined respiratory cycles and periods of voluntary apnea in healthy young men in a sitting position the patellar tendon reflex was mechanically elicited continually.During voluntary apnea a persistence of regular reflex fluctuations was observed which had about the same frequency of the previous spontaneous respiration.An exact coordination or a correlation in integral numbers could be demonstrated between reflex-fluctuations and a controlled respiration in intervals of 2.5–12 sec. When such a coordination had occured and an apnea was interposed, the reflex-fluctuations took a frequency of about 12/min like that of the previous spontaneous respiration independently of the frequency of the preceding controlled respiration.In the range of frequencies from the respiratory frequency to half that value a synchronisation between respiration and reflex fluctuations ensued immediately. During a controlled respiration with an interval between the spontaneous respiratory interval and double that interval, it took a longer time until an exact coordination of reflex fluctuations and respiration appeared. In addition, during apnea and extremely slow respiration coordinations of the tested parameters in a 1:2 or 1:3 ratio occurred.In comparison with earlier results of animal experiments conclusions are drawn concerning the possible site of the observed somato-vegetative coordinations.This investigation was supported by the Deutsche Forschungsgemeinschaft.     

Investigations into the origin of mouse liver sinusoidal cells

Summary The possibility that liver sinusoidal cells are derived from the bone-marrow was investigated in chimeric mice. H2^k-positive bone-marrow cells from F₁ (B10.BR × B10.D2) hybrid mice were transplanted into irradiated H2^k-negative parental mice (B10.D2), and the liver examined immunohistochemically for the presence of H2^k-positive cells, with the help of an anti-H2^k monoclonal antibody. With the passage of time (from the fifth week onwards), increasing numbers of transplanted bone-marrow cells enter the liver sinusoids, undergo alteration in their shape, and remain there, probably replacing sinusoidal lining cells. DNA-synthesising cells in the sinusoids were observed, suggesting, in addition, local cell proliferation. The replacement of sinusoidal cells from bone-marrow was greatly accelerated after liver damage had been induced by sublethal doses of endotoxin (LPS), and proliferation was also enhanced after treatment with LPS. These results strongly suggest that the bone-marrow participates in the replacement of liver sinusoidal cells.Dedicated to Prof. Dr. W. Gerok on the occasion of his 60th birthday, March 27,1986     

Investigations into the skin-irritant properties of resiniferonol ortho esters

A series of esters were produced by partial synthesis from 9,13,14-orthophenylacetyl-resiniferonol. These compounds were tested for irritant effects by means of a mouse ear assay. All of the derivatives, including the parent compound, produced short-term inflammation of mice ears within 1–2 h and the effects did not persist for 24 h. This is in contrast to esters of structurally related tigliane diterpenes which produce a longer-term effect on mice ears. Highly potent irritants were synthesized which exhibited irritant doses 50% in the 0.0012–0.00021 nmol range. These esters were all substituted phenylacetates of the C-20 position of 9,13,14-orthophenylacetyl-resiniferonol. The meta or para positions of the phenylacetate moiety were substituted with electronegative groups for maximum activity. Compounds exhibiting substituted phenyl propionates at C-20 were not irritants in the test used.     

Investigations into the role of reovirus in the malabsorption syndrome

Malabsorption syndrome, defined by five criteria, could not be reproduced by oral inoculation of newly hatched chicks with six reoviruses isolated from six different cases. Passage in birds of four reoviruses with intestinal homogenates did not result in increased pathogenicity. In contrast, inoculation of complete infectious intestinal homogenate caused great weight loss, long lasting excretion of yellow-orange mucoid and wet faeces, increased plasma alkaline phosphatase activity, decreased carotene concentration and bone abnormalities. Malabsorption syndrome could not be reproduced with infectious intestinal homogenate comprising only reovirus and possibly other non-enveloped viruses after treatment with methanol or chloroform. Infectious homogenate made reovirus-free by incubation with anti-serum was as pathogenic as homogenate that had been treated the same way with broth and that still contained viable reovirus. While infectious homogenate was almost apathogenic for 3-day-old chicks, its pathogenicity for birds of this age was greatly enhanced by a pre-infection with reovirus immediately after hatching. Reovirus therefore may act as a trigger in the malabsorption syndrome. This enhancing effect, however, was not specific for reovirus; it was also achieved with an adenovirus. Vaccination of two groups of breeders with two different inactivated reovirus vaccines, resulted in effective transfer of antibody to the offspring, but did not protect the offspring against malabsorption syndrome.     

Evaluation of a method for weighing small tissue samples: Investigations into freezing and evaporation

Summary A method for weighing very small wet tissue samples with a view to estimating percentage water is described, involving the use of gold foil buckets weighed on an electrobalance. A comparison of precision of this method and that of an existing method is presented. The effect on percentage water of freezing the tissue has been investigated. The rate of evaporation of water from tissue in unsealed containers has been estimated and a relationship shown with the weight of the tissue. The rate of evaporation can be minimized by folding correctly.     

The aerobic-anaerobic transition: re-examination of the threshold concept including an electromyographic approach

Summary The surface electromyogram (EMG) from the vastus lateralis muscle and the metabolic and respiratory parameters were studied simultaneously during an incremental exercise in order to identify EMG signal modifications during the aerobic-anaerobic transition. Subjects performed an incremental test on the bicycle ergometer from an initial work load of 175 W to exhaustion by steps of 25 W. Ventilatory flow ( _E), oxygen uptake ( ) and carbon dioxide flow ( ) were recorded continuously. For lactate concentration determination, venous blood samples were collected during the final 30 s of each step. EMG signals were stored on magnetic tape. They were then converted into successive spectra to allow the study of EMG total power (PEMG) and mean power frequency (MPF) evolutions. A non linear increase in blood lactate reflected by a breaking point at 250 W was observed. A change in ratio occured at 275 W. PEMG value showed a non linear increase reflected by a breaking point at 275 W. MPF value increased from the first to the seventh step with a tendency to decrease at the last step. A great interindividual variance of EMG data was observed indicating the difficulty of correlating mean values of EMG parameters with mean values of blood lactate in order to explain sudden lactate increase by fast twitch fibre recruitment. However, comparison of individual EMG data suggests a progressive recruitment of fast twitch fibres as work load increases.     

Investigations into the genotoxic potential of loxtidine, a long-acting H2-receptor antagonist

Loxtidine, a potent, non-competitive histamine H₂-receptor antagonistwas evaluated for genotoxic potential using a range of short-termmutagenicity assays. Unequivocally negative results were obtainedin a Salmonella/plate incorporation assay and a liquid pre-incubationassay (using S.typhimurium strains TA1535, TA100, TA1537, TA1538and TA98), a fluctuation assay [using Escherichia coli strainsWP2, WP2 uvrA (R46) and 343/113 lys60 (R46)], a gene conversionassay (using Saccharomyces cerevisiae JD1) and a human peripherallymphocyte cytogenetic assay. All of these in vitro tests werecarried out in the presence and absence of rat liver S9 mix.In addition, the major metabolites of loxtidine in the rat werealso negative in the same range of microbial mutagenicity assays.Loxtidine was inactive in the mouse micronucleus test afteroral administration. The potential nitrosatability of loxtidinewas investigated using an expanded version of the WHO NitrosationAssay Procedure, and detectable quantities of mutagenic nitroso-specieswere not formed. The subsequent appearance of carcinoid tumourswithin the gastric fundus of rodents treated orally with loxtidinefor most of their natural lifespan, led to additional assaysbeing carried out on this compound to determine whether thetumorigenic effects were due to alternative mutagenic mechanisms.Negative results were obtained in an in vitro unscheduled DNAsynthesis assay using primary rat hepatocytes, and an assayfor spindle damaging agents using Muntjac skin fibroblasts.It can be concluded from these results that loxtidine is unlikelyto be a genotoxic carcinogen. The increase in carcinoid tumourincidence observed in rats and mice after loxtidine treatmentwas probably related to the prolonged achlorhydria producedby this potent unsurmountable histamine H₂-receptor antagonist.     

Investigations into the Euglena method for the assay of the vitamin B12 in serum

Details are given of a modified Euglena method for the assay of B₁₂ in serum. Serum in very small quantities is usually inhibitory to the growth of the Euglena. In general, this inhibition is most marked when the organism is growing prolifically. For estimating serum B₁₂ values, a standard curve is used from B₁₂ standards containing 0·01 ml. of serum of low B₁₂ content, so that growth in test serum solutions is compared with growth in serum-containing standards. The recovery of B₁₂ added to serum, incomplete when estimated from aqueous standards, is now complete and the variation in results from batch to batch is markedly reduced.     

Investigations into the molecular effects of single nucleotide polymorphism.

OBJECTIVES: DNA sequences are very rich in short repeats and their pattern can be altered by point mutations. We wanted to investigate the effect of single nucleotide polymorphism (SNP) on the pattern of short DNA repeats and its biological consequences. METHODS: Analysis of the pattern of short DNA repeats of the Thy-1 sequence with and without SNP. Searching for DNA-binding factors in any region of significance. RESULTS: Comparing the pattern of short repeats in the Thy-1 gene sequences of Turkish patients with ataxia telangiectasia (AT) with the "wild type" sequence from the DNA database, we identified a missing 8-bp repeat element due to an SNP in position 1271 (intron II) in AT-DNA sequences. Only the mutated sequence had the potential for the formation of a stem loop in DNA or pre-mRNA. In super-shift experiments we found that DNA oligomers covering the area of this SNP formed a complex with proteins amongst which we identified the proliferating cell nuclear antigen (PCNA) protein. CONCLUSION: SNPs have the potential to alter DNA or pre-mRNA conformation. Although no SNP-depeding formation of the DNA-protein complex was evident, future investigations could reveal differential molecular mechanisms of cellular regulation.     

Investigations into the free-living phase of the cat hookworm life cycle

Summary Laboratory studies were undertaken to evaluate the role of environmental factors on the development of free-living stages of the cat hookwormAncylostoma tubaeforme. An index of development calculated from the proportion of eggs that developed to the infective stage and the reciprocal of the development period, has suggested that the absence of high temperatures (26°–30°C) would significantly affect the L₃ population in contaminated soils.There were, however, no significant differences in the size and lipid content of L₃ that had developed at the different temperatures.     

Investigations into the Mass Spectrometric Method for the Determination of the Mode of Termination in Radical Polymerization

The fraction of termination by disproportionation, λ, in radical polymerization may be determined by a mass spectrometric (MS) analysis of the resulting polymer. It is subjected to an array of stringent consistency checks, working with polymerizations of methyl methacrylate at 85 °C in three different solvents and with four different initiators. λ is shown to be unaffected by the choice of initiator, initiator concentration, or isoviscous solvent. These findings serve to allay any fears about the method being undermined by effects such as primary radical termination or chain transfer to solvent, thereby establishing its robustness. At the same time, direct evidence for the occurrence of chain transfer to the solvent methyl isobutyrate is uncovered, and the importance of knowing other rate‐parameter values accurately, if λ is to be determined accurately, is illustrated. By carrying out MS analyses, it is concluded that electrospray ionization with time‐of‐flight detection gives the best results for the present purpose.

     

Investigations into the mechanism of progesterone release by rat lutein cells

Vinblastine treatment blocks progesterone release from the lutein cells of superovulated immature rat ovaries, without impairing hormone synthesis, and induces the accumulation of electron-dense granules, 0.2--0.3 micron in diameter, at the juxta-sinusoidal poles of the cells. The hypothesis is advanced that these granules are true secretory organelles and that an exocytotic mechanism underlies the progesterone release from rat lutein cells.