Etiological factors and mechanism involved in relationships between pesticide exposure and cancer.

Professional as well as public exposure to pesticides raise cancer risk. Interaction with adjuvants and with other toxicants increases the actual risk. Endocrine disruption, procarcinogen activation by detoxification enzymes and intercellular communication impairment are involved in the carcinogenic processes. Organochlorine pesticides, including DDT, persist in human tissues for years, with correlated breast cancer incidence. One major point usually underestimated in risk evaluation is the individual variability in detoxification capabilities. Furthermore cellular sites of cancerisation are not necessarily identical to sites primarily exposed to toxicants. Post-diagnostic stress factors and iatrogenic effects of treatment concur to complication of the status of patients. The abnormal increase in the rates of cancer emergence denotes a failure in the application of the precautionary action principle.

Coevolutionary toxicity as suggested by differential coniferyl alcohol inhibition of ceratocystis species growth.

Coevolution has been shown to lower the toxicity of predator venoms to usual preys, in contrast to higher toxicity to non-prey similar species (Heatwole and Powell, 1998. Resistance of eels (Gymnothorax) to the venom of sea kraits (Laticauda colubrina): a test of coevolution. Toxicon 36, 619-625). In an attempt to examine whether such coevolutionary discrepancies also occur in plant host-parasite interactions, two strains of Ceratocystis grown on artificial medium, C. fimbriata, parasite of the plane tree, and C. bruneociliata, parasite of the Scots pine (Pinus sylvestris), were compared for growth parameters, in controls and in presence of various concentrations of coniferyl alcohol (a phenolic derivative previously found to be released following inoculation of pine trees with C. bruneociliata). Coniferyl alcohol differently inhibited the growth of both fungi. In the case of the conifer-specific fungus, inhibition rate was less marked at low doses (<2.5 mM) but it rose more steeply at higher doses (10 mM) after a sigmoidal transition at around 3.2 mM, indicating a physiological threshold. These results support the hypothesis of a specific action of coniferyl alcohol against C. Bruneociliata, as a coevolutionary adaptative characteristics of the fungus.

Toxicity of cypermethrin and fenitrothion on the hemolymph carbohydrates, head acetylcholinesterase, and thoracic muscle Na+, K+-ATPase of emerging honeybees (Apis mellifera mellifera. L).

Comparative effects of sublethal doses (0, 0.1, 0.2. 0.4, 0.8, and 1 nmol/bee) of cypermethrin and fenitrothion have been studied on emerging honeybees. The insecticides were injected intrathoracially between the third and the fourth segment. Biochemical effects were determined over a 3-h period. Both cypermethrin and fenitrothion led to a significant hypoglucosemia and hypotrehalosemia 15 min after injection, but cypermethrin seemed more active than fenitrothion at the same doses. A recovery phase appeared for glucosemia and trehalosemia, 60 min after injection. The higher toxicity of cypermethrin than fenitrothion also appeared in this period, where it took a longer time for honeybees to reestablish carbohydrate levels following cypermethrin than fenitrothion injections. The low values of the correlation coefficients (r) for glucose versus trehalose levels led to the supposition that no typical functional interaction between glucose and trehalose could be considered to be involved in this experience. Na+, K+-ATPases activity was significantly inhibited (P< 0.05) by cypermethrin and maximum percentage inhibition was reached (45%) at 1 nmol/bee. The kinetic analysis of honeybee's acetylcholinesterase inhibition by fenitrothion, indicated that this insecticide acts (P< 0.05) on acetylcholinesterase activity. The percentage inhibition exceeded 60% at 0.2 nmol/bee. This result revealed that in general cypermethrin and fenitrothion share common biochemical effects on carbohydrates, although their neurotoxic effects on honeybees might be different.

Biological and biochemical effects of chronic exposure to very low levels of dietary cypermethrin (Cymbush) on honeybee colonies (Hymenoptera: Apidae).

Cymbush (100 g of pure cypermethrin/liter of petroleum ether) was added to sucrose syrup at 12.5 microg/L and given to honeybee colonies (Apis mellifera mellifera, L.) in their diet for 5 months (July to November). Many perturbations have been recorded in treated groups in contrast to controls placed in the same area. Mortality in the hive, bee behavior, brood areas, supersedure, glucosemia, trehalosemia, and (Na+,K+)ATPase activity are many factors significantly affected over the 18-week test following sublethal treatment. Results suggested that long-term exposure of honeybees to cypermethrin-contaminated diets at concentrations not immediately lethal to worker adults may cause significant hidden damage to colonies.

Biochemical effects of high intensity constant magnetic fields on worker honey bees.

Hemolymph samples from adult bees that had completed their pupal development and emergence in a 7 Tesla field contained a lower percentage of glucose than controls, indicating that trehalase enzyme activity in honey bees is reduced in strong magnetic fields. Significantly more phospholipids were found in the intestines of magnetic field-exposed bees than in controls. No significant differences were found for fatty acids, triacylglycerols, or steroids.

Worker honeybee hemolymph lipid composition and synodic lunar cycle periodicities.

The aim of the present investigation was to extend a previous study, showing a correlation of the variations of hemolymph carbohydrates with synodic lunar-like cycle and its circaseptan harmonics to worker honeybee hemolymph lipids. Hemolymph lipid concentrations of emerging worker imagos were analyzed in terms of one ideal synodic lunar cycle and processed by the cosinor method testing the null hypothesis versus the presence of 29.5-, 14.8- or 7.4-day periods in the data. A rhythmicity statistically compatible with a 29.5-day rhythm was observed for triacylglycerols and steroids as well as for body weight. A circadiseptan rhythm was determined for 1,3 diacylglycerols, while fatty acids and phospholipids exhibited a circaseptan rhythm. An agreement of peaks for triacylglycerols, steroids and body weight at the new moon, but not at the full moon, was noted with respect to trehalose and glucose circadiseptan rhythms. The latter moon-phase timing of peaks and nadirs, compared with that previously determined for trehalose and glucose, appeared to be identical to the circadiseptan rhythm and reciprocal for the circaseptan rhythms of 1,3 diacylglycerols. Reciprocal tendencies in circaseptans of trehalose and glucose on the one hand, and fatty acids and phospholipids on the other are indicated. The underlying causal nexus of these relationships is unknown.

The topology of perceptive functions as a corollary of the theorem of existence in closed spaces.

The capability for a system of perceiving both outer objects and an inner self are two fundamental features of abstract mathematical objects endowed with the properties of topologically closed sets. Such structures exist upon intersection of topological spaces owning different dimensions. Then, the theorem of Jordan-Veblen provides their capability of being observable, while the theorems of Brouwer and of Banach-Caccioppoli provide two kinds of fixed points which account for the properties of so-called right and left brain functions. Fixed points account for the biological 'self', and the system provides theoretical justification for the existence of brain structure/function relationships, including memory, emotion, and respective characteristics of right and left hemispheres. Hence, an abstract topological reasoning based on set properties, provides evidence that the observer's function directly infers from the phenomenon of existence and that it belongs to the same mathematical system as the property of being observable. Order relations are raised from equivalence relations by Poincaré groups, upon mappings on the sets of functions and related homotopic transformations in sequences of intersections. Therefore, time is a construction of abstract brain functions, and a living organism just fills the system with appropriate molecular structures.

Effects of catechols on free radical formation by chemotherapeutic agents (adriamycin, farmorubicin, and mitomycin).

The aim of the present study was to investigate the effects of biologically important catechols on the cytotoxicity of adriamycin, farmorubicin, and mitomycin C with respect to hydroxyl radical production. Catecholamines (adrenalin, noradrenaline, dopamine) and DOPA enhance the generation of hydroxyl radicals by chemotherapeutic antibiotics. Measures were done using a deoxyribose assay, in presence of the Co(II) + H2O2 system. Catalase and hydroxyl radical scavengers (mannitol, thiourea, cysteine, glutathione, L-lactic dehydrogenase) inhibited the deoxyribose damage caused by the drugs.

Toxicology of cupric salts on honeybees. V. Gluconate and sulfate action on gut alkaline and acid phosphatases.

Some aspects of putative nontarget effects of cupric ions systemically fed to honeybees against their parasite mite Varroa jacobsoni have been investigated on the host phosphatases. The alkaline and acid forms extracted from the guts of worker bees exhibited substrate-inhibition features. Upon detailed kinetic analysis, cupric organic salts indicate activation effects at concentrations of about 1 mM. Concentrations up to 10 mM (alkaline form) and 25 mM (acid form) induced no important changes, except a partial quenching of the substrate-inhibition process, characterized by a wide increase in the constant of apparent inhibitory binding of substrate to the enzyme-substrate complex. Partial purification gave a single alkaline form with quite similar kinetic behavior in the absence of natural ions as in crude extracts. Cupric gluconate and sulfate demonstrated similar patterns, except an increase of the apparent Hill coefficient by sulfate only. The substrate constant of acid phosphatases was decreased at high cupric gluconate doses while its maximum velocity was biphasically increased (with observed maximum at 1 mM), resulting in a sustained activation. Chemiluminescence studies revealed that cupric ion activation is counteracted by oxygen radicals generated by cupric ions and also, in vitro, by the artificial substrate para-nitrophenylphosphate. The para-nitrophenol molecules released from the reaction are therefore responsible for biphasic effects selectively observed with gluconate salts. In apicultural practice, neither blockade of activity nor dramatic changes are to be expected at doses administered to bees against the parasite.

Statistical approach for evolutionary relationship of alpha-conotoxins and members of insulin-superfamily.

Similar conserved structures appear in apparently unrelated protein families. Thus, the superfamily of insulin shows an evolutionary relationship with the alpha-conotoxins of marine fish-hunting snails as indicated by methods of protein comparison. In order to reach statistical significance, the A-chains of different insulins, insulin-like growth factors, relaxins, insulin related peptides from invertebrates were drawn for comparison. These data were correlated with sequences from randomly chosen proteins. The alpha-conotoxins show identity scores up to 37.5% and similarity up to 56.2% toward the members of the insulin-superfamily. These scores conform to values achieved by comparing the relaxin and the insulin/IGF-sequences. The data show clearly that the identity and similarity values obtained in the comparison with the insulins are significantly higher than the scores of randomly chosen protein primary structures. According to our calculated data, this hormone system regulating metabolism and growth in vertebrates and the mentioned toxin-receptor system share the same evolutionary ancestor. However, this statistical approach has to be substantiated on gene level.

Beware of a new carcinogenic risk: promotion of promoters.

Restricting the evaluation of carcinogenic risk to genotoxicity or mutagenicity tests, as recently recommended by some researchers, is a hazardous operation, since the carcinogenic process involves a complex sequence of events in which no simple, necessary and sufficient condition is known for either the occurrence or non-occurrence of a cancer. None of the available tests exhibit a generalizable meaning, while none of the carcinogenic process phases should be neglected in prediction studies. In particular, ignoring promoter action and progression or proliferation, as well as targets specificity and other points, should lead to an underestimation of the actual risk.

Toxicology of cupric salts in honeybees. I. Hormesis effects of organic derivatives on lethality parameters.

Feeding bees with organic cupric salts provides long-term control of the parasite Varroa jacobsoni. A set of new algebraic parameters (M. Bounias C.R. Acad. Sci. 310(3), 65-70, 1990) completely describing the population lethality function has been calculated following chronic administration of cupric gluconate, aspartate, and isoleucinate, with or without dietary pollen. Mortality curves allowed the calculation of LT50 (time for 50% lethality) as well as Hill coefficients (h) of the curves and the LD50 as a function of time. The tangent at the inflexion point of the sigmoidal time/mortality curves (delta i) gave the maximum mortality acceleration as an additional parameter. No toxicity (i.e., no decrease of TL50 vs doses and no LD50 values) was found for cupric gluconate and isoleucinate with pollen, whereas increases in LT50 and decreases in delta indicated hormesis effects. Doses decreasing by half-time LT50, h, or delta were used as objective lethality indexes for comparisons of toxicity in the other cases. Routine acute toxicity at high dosage was also compared with phosalone and lindane effects 24 hr after treatment.

Toxicology of cupric salts on honeybees. IV--Gluconate and sulfate action on hemolymph trehalose activity in vivo and in vitro.

A biphasic increase of hemolymph glucose levels was observed following injection to bees of cupric gluconate or sulfate, both potent agents for the control of Varroa jacobsoni, a parasitic mite of hives. The simultaneous injection to bees of 0.3 microM BAYg5421 (an inhibitor of alpha-glucosidases) quenched the response, suggesting a direct effect of 2 nmol/bee cupric ions on trehaloses' activity. One nanomol of injected cupric gluconate increased the trehalose (Tre) activity by 233% in crude hemolymph extracts at 1 mM trehalose concentration, and exhibited biphasic dose-related effects with a maximum 15% increase at 0.5 mM cupric ion and a stabilized 20% inhibition from 4 mM, regardless of the anionic moiety. Upon partial purification of the enzyme complex, two fractions (FI = 75% and FII = 25% of total activity) were isolated that exhibited, respectively, less and more marked positive cooperatively than crude extract. Form I showed almost no susceptibility to either cupric derivatives, which indicated form II as the most likely target, with 68% and 72% increases with 0.25 mM cupric sulfate and 0.5 mM cupric gluconate, in presence of 16 mM trehalose.

Molecular activation of a trehalase purified from the fat body of a coleopteran insect (Tenebrio molitor), by an endogenous insulin-like peptide.

Trehalase is the major factor involved in the release of glucose in the various insects organs. During physiological regulatory processes, and particularly during the induction or breaking of diapause, insulin-like factors are involved. Then, by contrast with the classical hypoglycemic role of insulin, the insulin-like peptide isolated from the insect (brain and/or fat body) is also able to activate the fat body trehalase in vitro, through a direct molecular interaction. The mechanism involves the binding of the activator to the "trehalase-trehalose complex", rather than on the free enzyme, without change in the Hill coefficient nor in the maximum velocity (Vmax). The rate constant KR is conversely proportional to the activator concentration. This mechanism can be totally quenched by adding an anti-serum anti-insulin-like factor in the reaction medium.

Time and dose-related effects of the pyrethroid fluvalinate on haemolymph carbohydrates and gut lipids of honeybees, following in vivo injection of very low doses.

The injection to emerging adult workerbees with fluvalinate doses ranging from 1 femtomol to 1 nanomol per individual resulted in a reduction of haemolymph carbohydrate concentrations, particularly at the lowest dose 1 hour after injections. At the same time, a large increase was observed for triacylglycerols and to a much lesser extent for steroids and phospholipids with 0.1 picomol per bee. By contrast, fatty acids, steroids and triacylglycerols exhibited a depress at the higher dose. Most responses were thus biphasic, showing that much attention should be paid to the effects of very low doses of pesticide.

Fluctuations of carbohydrates in haemolymph of honeybee (Apis mellifica) after fasting, feeding and stress.

The haemolymph of honeybee contains trehalose, a non-reducing disaccharide, glucose and fructose. The aim of this work was to describe changes of haemolymph sugar induced by different physiological situations. Gas chromatography for the determination of carbohydrate concentrations in the haemolymph was introduced. The individual bee was anesthetized by CO2 and punctured at tergum III in order to gain 1 microliter haemolymph using a glass capilette. The main sugars in the haemolymph were determined as silylated derivatives by gas chromatography. Bees living under exactly standardized conditions in an artificial bee-house were observed after feeding and fasting. In addition saccharides were determined after "run-stress" along different distances.

Changes in the properties of honeybee haemolymph alpha-glucosidases following dithiothreitol dissociation of native complexes.

1. The higher relative molecular mass (M(r)) forms of larval honeybee haemolymph alpha-glucosidases are dissociated by dithiothreitol (DTT) into lower M(r) electrophoretic forms, without any important loss of activity. 2. The maximum velocity remains unchanged and the apparent dissociation constant is slightly increased, with Ki approximately equal to 247 mM and I50 approximately equal to 730 mM. 3. By contrast, the major changes affect the Hill coefficient which decreases from 1.0 in controls to 0.7 in presence of 600 mM DTT. 4. In the absence of both DTT and substrate, the major native enzyme form, isolated by gel filtration, spontaneously rearranges to give three additional minor forms, one of lower M(r) and two of higher M(r). 5. These data are consistent with the hypothesis of substrate-directed aggregation of enzyme protomers into functional complexes.

Sublethal effects of the formamidine amitraz on honeybees gut lipids, following in vivo injections.

The time-course and dose-related action of amitraz (AMZ) on gut lipids of worker honeybees were examined over 3 hours following in vivo injections of 0.25, 0.5, 1 and 2 nmols of pesticide per bee. Significant decreases were observed at 30-45 min with 0.25 nmols per bee, for phospholipids, fatty acids, steroids and triacylglycerols. However increases were observed either later or with higher doses. The decreasing action observed with 0.25 nmol AMZ per bee was inhibited by simultaneous injections of the alpha antagonist phentolamine (from 0.25 to 2.0 nmols per bee). The toxicity of AMZ to honeybees thus likely involves the mobilization of lipids from the gut, via action of this formamidine pesticide on alpha-adrenoceptors.

[Kinetic study of mixed alpha glucosidase activity induced in honeybee hemolymph by in vivo injection of trehalose]. / Etude cinétique d'une activité alpha-glucosidasique mixte induite dans l'hémolymphe d'abeilles par injection de tréhalose in vivo.

In vivo injection, into emerging bees, of a quantity of trehalose sufficient to double the initial natural haemolymph level of the sugar, leads first to a reduction in the maximal velocity (Vm) of the alpha-glucosidase activity, after 2 hours, followed by an increase in Vm at 6 hours, then a return to normal. The substrate constant (KR) shows minor and insignificant fluctuations about the initial value, and the Hill coefficient (h) a slight, but significant, rise 2 h and 8 h after injection. The simultaneous injection of a protein synthesis inhibitor (ISP) reduces the changes in Vm and h, and lowers value of the ratio Vm/KR from 6 h to 16 h after injection. The overall pattern of results prompts the hypothesis of an intermediary ISP-sensitive induction of an inhibitor of the enzyme-substrate complex, preceding the phase of induction or liberation of active enzyme molecules whose aggregation is subject to a succession of rearrangements alternately favourable to sucrose (lesser specificity) or to trehalose (heightened specificity).

Glucagon as a non species-specific regulator of the glycaemia in honeybee (Apis mellifica).

It is known that hormones which exist in vertebrates are very old in respect to evolution. The presence of insulin and glucagon in insects had already been suggested in the early sixties. In this study honeybees are living in an "artificial hive" in the German laboratory under exactly standardized conditions. In the French laboratory standardized conditions yielded because only emerging worker bees were used i.e. those which were just slipping from the honeycomb alveoli. It seemed of interest to introduce an in vivo model in which one animal is punctured at tergum III in order to gain 1 microliter hemolymph using a glass capillary tube . *For the first time it is possible to puncture one animal three times. The main sugars in the hemolymph were silylated* and determined by gas chromatography in the German resp. by nano thin layer chromatography in the French laboratory. 0-500 ng porcine glucagon resp. the same amount of purified bee glucagon were injected to the animals and the action on carbohydrates observed during 2 hours. Similar to the action of glucagon in vertebrates, carbohydrate metabolism although completely different, appears to be regulated in part in this invertebrate stem. It is concluded that glucagon may represent a key hormone for degradation of the glycogen stores.