Loyd D. Rowe

Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity.

Abstract Monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of principally sodium ions for protons. The outer surface of the ionophore-ion comples is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes in nonpolar solvents. In biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. The net effect for monensin is a trans-membrane exchange of sodium ions for protons. However, the interaction of an ionophore with biological membranes, and its ionophoric expression, is highly dependent on the biochemical configuration of the membrane itself. One apparent consequence of this exchange is the neutralization of acidic intracellular compartments such as the trans Golgi apparatus cisternae and associated elements, lysosomes, and certain endosomes. This is accompanied by a disruption of trans Golgi apparatus cisternae and of lysosome and acidic endosome function. At the same time, Golgi apparatus cisternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions. Monensin effects on Golgi apparatus are observed in cells from a wide range of plant and animal species. The action of monensin is most often exerted on the trans half of the stacked cisternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensin concentrations or short exposure times, near the middle of the stacked cisternae. The effects of monensin are quite rapid in both animal and plant cells; i.e., changes in Golgi apparatus may be observed after only 2–5 min of exposure. It is implicit in these observations that the uptake of osmotically active cations is accompanied by a concomitant efflux of H+ and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of cisternae observed in electron micrographs. In the Golgi apparatus, late processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin. Yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the Golgi apparatus. In endocytosis, monensin does not prevent internalization. However, intracellular degradation of internalized ligands may be prevented. It is becoming clear that endocytosis involves both acidic and non-acidic compartments and that monensin inhibits those processes that normally occur in acidic compartments. Thus, monensin, which is capable of collapsing Na+ and H+ gradients, has gained wide-spread acceptance as a tool for studying Golgi apparatus function and for localizing and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. Among its advantages are the low concentrations at which inhibitions are produced (0.01–1.0 μM), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or ATP levels) and a reversible action. Because the affinity of monensin for Na+ is ten times that for K+, its nearest competitor, monensin mediates primarily a Na+-H+ exchange. Monensin has little tendency to bind calcium. Not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed in cattle. The mechanisms by which monensin interact with coccidia and rumen microflora to achieved these benefits are reasonably well documented. However, the interactions between monensin and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensin poisoning are well known. Equine species are particularly susceptible to monensin poisoning, and a common effect of monensin poisoning is vacuolization and/or swelling of mitochondria in striated muscle. Other pathological injuries to striated muscle, spleen, lung, liver and kidney also have been noted. A consistent observation is cardiac myocyte degeneration as well as vacuolization. Differences in cellular response resulting from exposure to monensin (i.e., Golgi apparatus swelling in cultured cells, isolated tissues, and plants vs.mitochondrial swelling in animals fed monensin) suggest that myocardial damage is due either to a monensin metabolite or is a secondary response to some other derivation. However, as pointed out by Bergen and Bates [26], the underlying mode of action of ionophores is on transmembrane ion fluxes which dissipate cation and proton gradients. Consequently, some or all of the observed monensin effects in vivo in animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes.

Linear furocoumarins and other constituents from thamnosma texana

Abstract Eight linear furocoumarins and three coumarins were isolated and identified from Thamnosma texana . They were xanthotoxin, imperatorin, bergapten, alloimperatorin methyl ether epoxide, heraclenin, isopimpinellin, psoralen, oxypeucedanin, and the coumarins herniarin, osthol and thamnosmin. The linear furocoumarins appear to be agents that account for the known photosensitizing properties of Thamnosma texana , and consequently its colloquial name, ‘blisterweed.’ This is the first report on the occurrence of imperatorin, heraclenin, oxypeucedanin, herniarin or osthol in any Thamnosma species.

Lead poisoning in cattle: Reassessment of the minimum toxic oral dose

After feeding male Holstein calves Pb acetate by nurse bottle it was found that daily Pb intakes of 2.7 mg Pb/kg can kill calves on milk diets in 20 days or less while 5.0 mg Pb/kg/day consistently caused signs of intoxication and death in 7 days. Absorption rate of Pb was rapid and tissue depositions were high in calves on milk replacer diet. Tissues were analyzed by atomic absorption spectrophotometry. Data suggest that diet, dosing method, and dosing time must be carefully considered in evaluations of minimum toxic dose. The consistent production of seizures at these low daily Pb intakes suggests that this calf model may be valuable in the study of Pb encephalopathy. (JMT)

The influence of milk diet, grain diet, and method of dosing on lead toxicity in young calves☆☆☆

Twenty 2- to 3-week-old calves were fed a milk diet or a mixture of grain and hay. After acclimatization, all calves were dosed po with lead acetate for 7 days, either in solution via a nursing bottle or in a gelatin capsule. At the end of the treatment period, 6 of 10 calves fed milk were either dead or showed multiple signs of Pb poisoning. All calves fed grain and hay appeared normal. Tissue Pb was significantly higher in calves on a milk diet (p less than 0.05) compared to tissue from calves on a grain and hay diet. No significant differences wee found when dosing methods wee compared. These data clearly show that diet but not dosing methods greatly influenced the absorption and tissue distribution of Pb in calves. delta-Aminolevulinic acid dehydratase (ALAD) activity in blood dropped to approximately 20% of pre-Pb exposure concentrations within 24 hr after the initial dose of Pb and remained at this concentration despite continued Pb administration. Blood Pb concentrations indicate that, in this model, the Pb enters the blood rapidly and retention is prolonged. The calf model warrants further studies on absorption and metabolism.

Hypocholesterolemia and hypocortisolemia in acute and terminal Babesia bovis infection

Sera from Babesia bovis-infected cross-bred cows and purebred steers were evaluated throughout the course of acute Babesia bovis infection for cholesterol and cortisol concentrations. Cholesterol and cortisol values of surviving cattle became markedly reduced (less than 50% of normal values) during the acute phase of the disease, Days 10-15 postinoculation, and returned to essentially normal values by the end of the 35 day experimental period. The combined control group values were approximately 2.98 mM/l cholesterol and 0.085 mM/l cortisol throughout the experimental period. The results suggest that if normal synthesis of cholesterol by hepatocytes or other tissues is not maintained, then cholesterol-derived compounds such as cortisol may not be produced at levels adequate to protect against the effects of stress and cell damage during the disease process.

Detection of coccidiostats by immunoassay

Coccidiosis is a complex disease that affects many farm animals, but which can be especially serious in poultry. Control of coccidiosis is essential for efficient, modern poultry productions; thus coccidiostats are extensively used to control this disease. The most commonly used coccidiostats in poultry are the polyether monocarboxylic ionophores including monensin and salinomycin. Polyether ionophores also improve feed utilization by ruminants. However, the quantity of such ionophores for effective control of coccidiosis in chickens may prove highly toxic to other agriculturally important animals. Over the past few years, we have developed monoclonal antibody‐based immunoassays for both salinomycin and monensin. In addition, we have developed immunoassays for dimetridazole and for halofuginone, the latter being a heavily used non‐ionophore coccidiostat. The advantages of the immunoassays over the conventional analytical methods are discussed as well as the characteristics of the individual immunoassays.

The effects of naltrexone on cadmium-induced increases in oral ethanol self-administration

Adult male rats were exposed to a standard laboratory diet (N = 20), or an adulterated diet containing 100 ppm added cadmium (N = 20), for 60 days. On Day 61, half the animals from each dietary condition received subcutaneous implants of two 30 mg naltrexone pellets, and the remaining half the animals received identical implants of 30 mg placebo pellets. One week later, animals from groups created by this interaction (Groups Control-Placebo, Control-Naltrexone, Cadmium-Placebo, Cadmium-Naltrexone) were tested in an ethanol self-administration paradigm that presented a 10% ethanol solution (v/v) in both a choice and nonchoice format. The results indicated that cadmium exposure increased the oral self-administration of ethanol in the choice setting where water was offered as an alternative, and the opiate antagonist naltrexone failed to attenuate this effect.

Detection of Phototoxic Activity in Plant Specimens Associated with Primary Photosensitization in Livestock using a Simple Microbiological Test

Ingestion of mushrooms is infrequently observed by owners and may be omitted from the initial history. When mushroom poisoning is suspected on the basis of histopathological lesions, additional history and tests may help to establish the diagnosis. Collection and identification of mushrooms from the animal’s environment will establish the types of mushrooms that the dog was potentially exposed to. Examination of vomitus, gastrointestinal contents, and/or feces for mushroom spores may provide definitive evidence of mushroom ingestion and possible identification of the genus and species. In human patients, early diagnosis is occasionally achieved by detection of amanitins in urine or other body fluids.” Due to delayed onset of clinical signs, patients usually receive only supportive and palliative treatment. The treatment for Amanita mushroom poisoning has recently been reviewed.” Unless treatment is initiated promptly after exposure and before significant amounts of toxin are absorbed, the final outcome of the case is dependent on the amount and type of mushroom ingested.

Lactose and milk replacer influence on lead absorption and lead toxicity in calves

The absorption, tissue deposition, retention, and excretion of ingested lead is in large part due to associated dietary factors. Young suckling calves are extremely susceptible to low doses of lead, especially when maintained totally on milk. Unfortunately, the complexity of milk makes it difficult to determine which constituent is actually responsible for increased Pb absorption. Recent studies have shown that lactose, the major carbohydrate of milk, is a dietary factor that increases the absorption of several minerals including Pb in rats. The authors laboratory has recently demonstrated that milk greatly increased the tissue deposition of lead in calves. Lactose, however, has not been considered in the ruminant animal. Moreover, liquid milk seems to increase the absorption of lead more significantly than powdered milk. The purpose of this study was to assess the influence of lactose and powdered milk on lead uptake and tissue distribution in calves.

Facile Synthesis of a Carboxylic Linker on Halofuginone: A Method to Derivatize Haptens Containing Both Amine and Hydroxy Functionalities

Abstract A succinic acid derivative of halofuginone was synthesized that could be easily bound to carrier proteins for halofuginone-specific antibody production. N-trimethylsilylimidazole was used to protect the hydroxyl group on halofuginone. Hydrolysis after treatment with succinic anhydride afforded the required derivative.