Genetic factors in neurotoxicology and neuropharmacology: A critical evaluation of the use of genetics as a research tool

Animals have evolved a detoxication system to enable them to survive in a hostile chemical environment in which foods contain many non-nutrient chemicals. Detoxication depends on enzymes which are often genetically polymorphic. As a result, inter-individual variation is common, and in humans several Mendelian loci have been identified. However, most variation in response is probably due to the action of several genes. Genetic variation in response to the neurotoxin MPTP and to chemically and physically-induced seizures is reviewed. In the former case, differences between pigmented and white mouse strains have been noted which are consistent with the hypothesis that humans are more sensitive than mice or rats because of the presence of melanin in human brains. However, variation in sensitivity probably also depends on other genes. In the case of audiogenic seizures, a single locus has been identified and mapped, but its relationship with seizures induced by other agents is not clear. Genetic variation in response to alcohol is also discussed. The failure of most toxicologists to consider genetic variation as a potentially confounding variable, and as a powerful research tool, is discussed critically in relation to non-repeatability of research on the neurotoxic effects of lead, and in relation to the genetic variation in MPTP, seizures, and alcohol response already noted. It seems clear that genetic methods provide a powerful research tool which is largely being ignored by toxicologists.     

Decrease of mRNA levels and biosynthesis of sucrase-isomaltase but not dipeptidylpeptidase IV in forskolin or monensin-treated Caco-2 cells

Treatment for 48 h of differentiated, confluent Caco-2 cells with 2.5 10^?5 M forskolin or 10^?6 M monensin, which produces a significant decrease of the de novo biosynthesis of sucrase-isomaltase, does not change quantitatively the de novo biosynthesis of dipeptidylpeptidase IV. Western blot analysis and silver nitrate staining indicate that neither drug induces any modification in the steady state expression of these two brush border hydrolases. Northern blot analysis shows that the level of dipeptidylpeptidase IV mRNA does not change in treated as compared to control Caco-2 cells. In contrast, forskolin and monensin dramatically decrease the level of sucrase-isomaltase mRNA. These observations suggest a separate regulation of biosynthesis for sucrase-isomaltase and dipeptidylpeptidase IV in intestinal cells. The mechanisms responsible for such a difference are discussed. Among them, the role of glucose metabolism, which is perturbed by both drugs, appears to be of crucial importance.     

Periodicity in fish otolith Sr,Na, and K corresponds with visual banding

Examination of a section of an otolith from a teleost fish by fast atom bombardment-secondary ion mass spectrometry (FAB-SIMS) revealed seasonal periodicity in Sr, Na, and K concentrations that corresponded with visually observed annual banding. Strontium maxima also corresponded with Na and K minima and vice versa. In addition there was a general, apparently age-related, trend in Sr levels, with concentrations at the edge of the otolith being higher than in the core.     

Inhibitory effects of flavonoids on several venom hyaluronidases.

In vitro studies showed that the flavonoid aglycones apigenin, luteolin and kaempferol inhibited the hyaluronidase activity of five different venoms dose-dependently. They were also able to delay the venom action when injected into mice. Naringenin, catechin and flavonoid glycosides had no effect. The flavonoids with unsubstituted hydroxyl groups at C-positions 5, 7 and 4', a double bond between carbons 2 and 3, as well as a ketone group at position 4, exhibited potent inhibitory actions on the venom hyaluronidases.     

Molecular basis of cardiotoxicity upon cobra envenomation

Various clinical manifestations leading to death have been documented in most cases of bites caused by venomous snakes. Cobra envenomation is an extremely variable process and known to cause profound neurological abnormalities. The complexity of cobra venom can induce multiple-organ failure, leading to death in case of severe envenomation. Intramuscular administration of Malayan spitting cobra (Naja sputatrix) crude venom at 1 g/g dose caused death in mice in approximately 3 h. Analysis of gene expression profiles in the heart, brain, kidney, liver and lung revealed 203 genes whose expression was altered by at least 3-fold in response to venom treatment. Of these, 50% were differentially expressed in the heart and included genes involved in inflammation, apoptosis, ion transport and energy metabolism. Electrocardiogram recordings and serum troponin T measurements indicated declining cardiac function and myocardial damage. This not only sheds light on the cardiotoxicity of cobra venom but also reveals the molecular networks affected during envenomation.Received 7 August 2004; received after revision 11 October 2004; accepted 4 November 2004     

Cyercenes,novel pyrones from the ascoglossan molluscCyerce cristallina. Tissue distribution,biosynthesis and possible involvement in defense and regenerative processes

The extraordinary regenerative phenomena in the marine ascoglossan molluscCyerce cristallina are described here for the first time. The possible correlation between cyercenes, pyrones recently isolated from the mollusc regenerating dorsal appendages (cerata), and the processes of chemical defense and regeneration in the mollusc, was investigated by studying the tissue distribution, biological activity and biogenesis of cyercenes. Differences in distribution between theC. cristallina mantle, cerata and mucous secretion were found. Cyercenes showed activity in theHydra vulgaris regeneration assay and the mosquito fish ichthyotoxicity assay. The de novo biosynthesis of cyercenes from propionic acid was demonstrated by means of in vivo adsorption experiments with radiolabeled propionate.     

The scorpine family of defensins: gene structure,alternative polyadenylation and fold recognition

Small cationic antimicrobial peptides (SCAMPs) as effectors of animal innate immunity provide the first defense against infectious pathogens. This class of molecules exists widely in invertebrate hemolymph and vertebrate skin secretion, but animal venoms are emerging as a new rich resource. Scorpine is a unique scorpion venom defensin peptide that has an extended amino-terminal sequence similar to cecropins. From the African scorpion Opistophthalmus carinatus venom gland, we isolated and identified several cDNAs encoding four new homologs of scorpine (named opiscorpines 1–4). Importantly, we show for the first time the existence of multiple opiscorpine mRNAs with variable 3 untranslated regions (UTRs) in the venom gland, which may be generated by alternative usage of polyadenylation signals. The complete opiscorpine gene structure including its promoter region is determined by genomic DNA amplification. Two large introns were found to be located within the 5 UTR and at the boundary of the mature peptide-coding region. Such a gene structure is distinct, when compared with other scorpion venom peptide genes. However, a comparative promoter analysis revealed that both opiscorpine and scorpion venom neurotoxins share a similar promoter organization. Sequence analysis and structural modeling allow us to group the scorpines and scorpion long-chain K-channel toxins together into one family that shares a similar fold with two distinct domains. The N-terminal cecropin-like domain displaying a clear antimicrobial activity implies that the scorpine family represents a group of real naturally occurring hybrids. Based on the phylogenetic analysis, a possible cooperative interaction between the N and C domains is elucidated, which provides an evolutionary basis for the design of a new class of anti-infectious drugs.Received 5 April 2004; accepted 17 May 2004     

Snake venom action: Are enzymes involved in it?

Summary Enzymes were the first clearly recognized components of snake venoms. When several more were discovered, attempts were made to correlate venom action with enzymic functions. The last few years have seen most successful efforts in the identification, isolation and structural elucidation of highly toxic polypeptides present in snake venoms, in particular of neurotoxins and membrane-active toxins. Following this development the polypeptides were called the true toxic components and the enzymes lost their previous central position in venom pharmacology. The time, therefore, has come to re-evaluate the role of enzymes in the complex interaction between snake and prey. While highly active polypeptides indeed dominate the action of hydrophiid venoms, they appear to play a lesser role in crotalid venom action as compared with enzyme components. Enzymes are involved in many levels of venom action, e. g. by serving as spreading factors, of by producing very active agents, such as bradykinin and lysolecithins in tissues of preys or predators. Some toxins, e. g. the membrane-active polypeptides appear to participate in the interaction between membrane phospholipids and venom phospholipases. The classical neurotoxin, -bungarotoxin, has been recognized as a powerful phospholipase. Several instances are known which indicate that some enzymes potentiate the toxic action of others; the analysis of a single enzyme may, therefore, not fully reveal its biofunction. For 3 enzymes, ophidianl-amino acid oxidase, ATPpyrophosphatase, and acetylcholinesterase, some of the problems pertaining to venom toxicity are discussed.     

A presynaptic effect of whole venom ofDendroaspis jamesoni on the hemidiaphragm of rat

Summary Dendroaspis jamesoni venom in a dose of 12.5 g/ml restricts the uptake of Ca⁺ leading to inhibition of release of Ach from the motor nerve terminals of the hemidiaphragms of rats.Acknowledgment. University of Nairobi, for the research grant (No. 670-052), which supported this work. We also thank Mr. S. K. Githaiga, Government Chemist's Department, for his help in the use of the spectrophotometer.     

Facile functional analysis of insect odorant receptors expressed in the fruit fly: validation with receptors from taxonomically distant and closely related species

With the advent of genomic sequences and next-generation sequencing technologies (RNA-Seq), multiple repertoires of olfactory proteins in various insect species are being unraveled. However, functional analyses are lagging behind due in part to the lack of simple and reliable methods for heterologous expression of odorant receptors (ORs). While the Xenopus oocyte recording system fulfills some of this lacuna, this system is devoid of other olfactory proteins, thus testing only the “naked” ORs. Recently, a moth OR was expressed in the majority of neurons in the antennae of the fruit fly using Orco-GAL4 to drive expression of the moth OR. Electroantennogram (EAG) was used to de-orphanize the moth OR, but generic application of this approach was brought to question. Here, we describe that this system works with ORs not only from taxonomically distant insect species (moth), but also closely related species (mosquito), even when the fruit fly has highly sensitive innate ORs for the odorant being tested. We demonstrate that Orco-GAL4 flies expressing the silkworm pheromone receptor, BmorOR1, showed significantly higher responses to the sex pheromone bombykol than the control lines used to drive expression. Additionally, we show that flies expressing an OR from the Southern house mosquito, CquiOR2, gave significantly stronger responses to the cognate odorants indole and 2-methylphenol than the “background noise” recorder from control lines. In summary, we validate the use of Orco-GAL4 driven UAS-OR lines along with EAG analysis as a simple alternative for de-orphanization and functional studies of insect ORs in an intact olfactory system.     

Effect of (+)-catechin on renal and intestinal transport

Summary The ability of dog renal cortex slices to accumulate -methyl-glucoside or glycine is enhanced by the flavonoid (+)-catechin at a concentration of 3.5 mM. This stimulatory effect is apparently due to a decreased rate of efflux of either substrate. On the other hand, the uptake of p-amino-hippuric acid and N¹-methyl-nicotinamide is inhibited by (+)-catechin. The drug at the same concentration is without action on amino-acid transport by guineapig intestine in vitro.This study was supported by grants from Zyma S.A., Nyon. We are grateful to MmesH. Capt andP. Ganguillet and MlleD. Mettraux for their skilful technical assistance.     

Dyslexia and specifically distorted drawings of the face—a new subgroup with prosopagnosia-like signs

Summary Identification of a subgroup (38%) of dyslexics (new syndrome?). These, against controls (5.5%), drew neolithic face configurations analogous to those visually experienced in prosop-agnosia. Essential symptoms of this subgroup are seen as result of specific early ways of processing visual data: lexical shapes (letters, words) and facial features, as if these were concrete entities, not abstract component parts. Thus, with letters, taken as entities, d=q, d=b, N=Z.This study was made possible by a visiting professorship at the University of Heidelberg, Department of Neurology. Thanks are due to its Director, Professor Dr.Heinz Gänshirt, as well as to DirectorSchmitt of the Heidelberger Schulamt and to Psychologist GretRuder-Trümpy for facilitating the collection of the drawings.