Uptake of 203Hg2+ in the olfactory system in pike

Inorganic mercury ²⁰³Hg²⁺ was applied to the olfactory chambers or was given i.v. to pike (Esox lucius) and the uptake of the metal in the olfactory system and the brain was examined by autoradiography and gamma spectrometry. Application of ²⁰³Hg²⁺ in the olfactory chambers resulted in an accumulation of the metal in the olfactory nerves and the anterior parts of the olfactory bulbs of the brain. The levels of ²⁰³Hg²⁺ in other brain areas, such as the telencephalon, the optic tecti and the cerebellum, remained low. Application of ²⁰³Hg²⁺ in only one olfactory chamber resulted in an uptake of the metal only in the ipsilateral olfactory nerve and olfactory bulb. Intravenous injection of the ²⁰³Hg²⁺ resulted in a labelling of the olfactory system and the brain, which was much lower than of the blood. These results indicate that the ²⁰³Hg²⁺ is taken up in the olfactory neurones from the olfactory receptor cells in the olfactory rosettes and is transported to the terminal parts of the olfactory neurones in the olfactory bulbs. The uptake of mercury as well as some other metals in the olfactory system may result in noxious effects and this may be an important component in the toxicology of metals in fish.     

Axonal regeneration of fish optic nerve after injury

Since Sperry's work in the 1950s, it has been known that the central nervous system (CNS) neurons of lower vertebrates such as fish and amphibians can regenerate after axotomy, whereas the CNS neurons of mammals become apoptotic after axotomy. The goldfish optic nerve (ON) is one of the most studied animal models of CNS regeneration. Morphological changes in the goldfish retina and tectum after ON transection were first researched in the 1970s-1980s. Many biochemical studies of neurite outgrowth-promoting substances were then carried out in the 1980s-1990s. Many factors have been reported to be active substances that show increased levels during fish ON regeneration, as shown by using various protein chemistry techniques. However, there are very few molecular cloning techniques for studying ON regeneration after injury. In this review article, we summarize the neurite outgrowth-promoting factors reported by other researchers and describe our strategies for searching for ON regenerating molecules using a differential hybridization technique in the goldfish visual system. The process of goldfish ON regeneration after injury is very long. It takes about half a year from the start of axonal regrowth to complete restoration of vision. The process has been classified into three stages: early, middle and late. We screened for genes with increased expression during regeneration using axotomized goldfish retinal and tectal cDNA libraries and obtained stage-specific cDNA clones that were upregulated in the retina and tectum. We further discuss functional roles of these molecules in the regeneration processes of goldfish ON.     

Harmful effects of cadmium on olfactory system in mice

The inhalation of certain metals can result in olfactory epithelial injury, an altered sense of smell, and direct delivery of the metal from the olfactory epithelium to the olfactory bulbs and other parts of the central nervous system. The purpose of this study was to examine whether mice given an intranasal instillation of cadmium would develop altered olfactory function and to assess whether cadmium may be transported directly from the olfactory epithelium to the central nervous system. To evaluate cadmium's ability to induce anosmia and on the basis of olfactory epithelium sensitivity to metals, the aim of this study was first to study cadmium effects on the olfactory function and secondly to check whether cadmium may be transported from the nasal area to the central nervous system. After an intranasal instillation of a solution containing CdCl2 at 136 mM, we observed in treated mice: (1) a partial destruction of the olfactory epithelium, which is reduced to three or four basal cell layers followed by a progressive regeneration; (2) a loss of odor discrimination with a subsequent recovery; and (3) a cadmium uptake by olfactory bulbs demonstrated using atomic absorption spectrophotometry, but not by other parts of the central nervous system. Cadmium was delivered to the olfactory bulbs, most likely along the olfactory nerve, thereby bypassing the intact blood-brain barrier. We consider that cadmium can penetrate olfactory epithelium and hence be transported to olfactory bulbs. The olfactory route could therefore be a likely way to reach the brain and should be taken into account for occupational risk assessments for this metal.     

Involvement of active transport systems in the mobilization of cadmium by dithiocarbamates in vivo.

Previously, we reported that the action of cadmium (Cd) complexing dithiocarbamates, such as N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD), in removing Cd from the kidney involves a probenecid-sensitive organic anion transport system. However, other mechanisms responsible for Cd mobilizing effects of BGD and HBGD are still unclear. Therefore, in the present study we examined the effects of phloretin (an inhibitor of plasma membrane glucose carrier), phloridzin (an inhibitor of Na(+)-dependent active hexose transport) and alpha-aminoisobutyric acid (AIB, an inhibitor of amino acid transport) on the excretion and distribution of the chelating agents and Cd in mice. Phloretin pretreatment markedly decreased the biliary and urinary excretions of BGD and HBGD. Phloridzin pretreatment also decreased the biliary and urinary excretions of HBGD, but had no effect on the BGD. AIB pretreatment had no effect on the excretions of either BGD or HBGD. Phloretin pretreatment increased the hepatic and renal contents of BGD and HBGD. Contrary to this, phloridzin pretreatment decreased the hepatic content of BGD and hepatic and renal contents of HBGD, while AIB pretreatment decreased the renal contents of BGD and HBGD. The mobilizing effects of BGD and HBGD on the hepatic and renal Cd was also investigated using Cd-exposed mice. Phloretin or phloridzin pretreatment decreased the mobilizing effect of BGD and HBGD on the hepatic Cd, but had no effect on the renal Cd. These results suggest that BGD and HBGD are taken up into the liver and kidney by phloridzin- and phloretin-sensitive transport system, respectively; that Cd-BGD and Cd-HBGD complexes formed in the hepatic cells are secreted to the bile by phloridzin- and phloretin-sensitive transport systems; and that free BGD and HBGD secreted from these organ to the bile and urine might have occurred, at least in part, by different mechanisms.     

Intranasal delivery of insulin via the olfactory nerve pathway

Objectives Intranasal delivery has been shown to target peptide therapeutics to the central nervous system (CNS) of animal models and induce specific neurological responses. In an investigation into the pathways by which intranasal administration delivers insulin to the CNS, this study has focused on the direct delivery of insulin from the olfactory mucosa to the olfactory bulbs via the olfactory nerve pathway. Methods Nasal and olfactory tissues of mice were imaged with fluorescent and electron microscopy 30 min following intranasal administration. Key findings Macroscopic analysis confirmed delivery to the anterior regions of the olfactory bulbs. Confocal microscopy captured delivery along the olfactory nerve bundles exiting the nasal mucosa, traversing the cribriform plate and entering the bulbs. With electron microscopy, insulin was found within cells of the olfactory nerve layer and glomerular layer of the olfactory bulbs. Conclusions These results demonstrated that intranasal administration of labelled insulin targeted the CNS through the olfactory nerve pathway in mice.     

Kinetics of cadmium transport in the isolated perfused rat liver

The uptake of cadmium by the isolated perfused rat liver obeys first-order kinetics at each dose level tested, 0.178, 1.78, and 17.8 μmol cadmium as the chloride. The uptake rate constant is elevated at low cadmium doses indicating the possibility of a mediated transport process. Zinc is secreted into the perfusion medium by the isolated liver at a constant rate in control preparations (3.5 ± 1.2 nmol/min); however, this rate is significantly increased during cadmium exposure in a dose-dependent manner. At low doses the amount of excess zinc secreted by the isolated liver in response to cadmium exposure is approximately equal to the amount of cadmium taken up, on a molar basis. The data are consistent with the hypothesis that cadmium-zinc exchange is one mechanism for mediated cadmium transport in the liver. Cadmium rapidly appears in the bile following exposure, and biliary concentrations increase in relation to dose. The fraction of the cadmium dose excreted in the bile during 3 hr of cadmium exposure reached maximum levels at intermediate doses (6.9% excreted), indicating a threshold for biliary clearance at the low doses and supression of biliary excretion at the high doses due to reduced bile production.     

Axonal transport of receptors: coexistence with neurotransmitter and recycling

Receptor sites move along the axon of neurones by means of fast transport mechanisms. In rat sciatic nerves, the accumulation of muscarinic receptors above a ligature was linear up to 24 hr after the operation and exactly paralleled that of dopamine-beta-hydroxylase. After 6-hydroxydopamine treatment, there was a marked reduction of these receptor sites in the rat sciatic nerve and the spleen. Fractionation of dog splenic nerves by differential and isopycnic centrifugation enabled us to show that the muscarinic receptors in sympathetic nerves are associated with noradrenaline and dopamine-beta-hydroxylase in synaptic vesicles. Receptor and neurotransmitter may thus coexist in the same subcellular organelle. Opiate receptors measured in vitro and in vivo with 3H-lofentanil in the rat vagus nerve were found to accumulate on both sides of the ligature. After capsaicin treatment this accumulation was markedly reduced suggesting that the opiate receptors in the vagus are mainly associated with substance P neurones. Double ligature experiments suggest the existence of a recycling phenomenon in the perikaryon for vesicles containing muscarinic receptors and dopamine-beta-hydroxylase. This may represent a pathway to convey informational molecules from the extracellular to the intracellular compartment of a neurone.     

Evaluating transport of manganese from olfactory mucosa to striatum by pharmacokinetic modeling.

Increased brain manganese (Mn) following inhalation can result from direct transport via olfactory neurons and blood delivery. Human health risk assessments for Mn should consider the relative importance of these pathways. The objective of this study was to develop a pharmacokinetic model describing the olfactory transport and blood delivery of Mn in rats following acute MnCl(2) or MnHPO(4) inhalation. Model compartments included the olfactory mucosa (OM), olfactory bulb, olfactory tract and tubercle, and striatum. Intercompartmental transport of Mn was described as ipsilateral, anterograde movement to deeper brain regions. Each compartment contained free and bound Mn and included blood influx and efflux. First-order rate constants were used to describe transport. Model parameters were estimated by comparing the model with published experimental data in rats exposed by inhalation to (54)MnCl(2) or (54)MnHPO(4) with both nostrils patent or one nostril occluded. The model-derived elimination rate constant from the OM was higher for the chloride salt (0.022 per hour) compared with the phosphate salt (0.011 per hour), consistent with their relative solubilities. Rate constants for Mn transport among the other compartments were similar for both Mn forms. Our results indicate that direct olfactory transport provided the majority of Mn tracer in the olfactory regions during the 21 days following exposure to (54)MnHPO(4) and 8 days following exposure to (54)MnCl(2). Only a small fraction of Mn tracer from the tract and tubercle was predicted to be delivered to the striatum, 3 and 0.1% following (54)MnHPO(4) or (54)MnCl(2) exposure, respectively.     

Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish

The toxic effects of cadmium and other metals have been well established. A primary target of these metals is known to be the olfactory system, and fish exposed to a number of different waterborne metals display deficiencies in olfaction. Importantly, exposure over embryonic/larval development periods can cause deficits in chemosensory function in juvenile fish, but the specific cell types affected are unknown. We have previously characterized a transgenic zebrafish strain expressing the green fluorescent protein (eGFP) gene linked to the hsp70 gene promoter, and shown it to be a useful tool for examining cell-specific toxicity in living embryos and larvae. Here we show that the hsp70/eGFP transgene is strongly and specifically upregulated within the olfactory sensory neurons (OSNs) of transgenic zebrafish larvae following a brief 3-h exposure to water-borne cadmium. This molecular response was closely correlated to an endpoint for tissue damage within the olfactory placode, namely cell death. Furthermore, cadmium-induced olfactory cytotoxicity in zebrafish larvae gives rise to more permanent effects. Juvenile zebrafish briefly exposed to cadmium during early larval development display deficits in olfactory-dependent predator avoidance behaviors 4-6 weeks after a return to clean water. Lateral line neuromasts of exposed zebrafish larvae also activate both the endogenous hsp70 gene and the hsp70/eGFP transgene. The data reveal that even a very brief exposure period that gives rise to cell death within the developing olfactory placode results in long-term deficits in olfaction, and that hsp70/eGFP may serve as an effective indicator of sublethal cadmium exposure in sensory cells.     

Evidence of active transport of cadmium complexing dithiocarbamates into renal and hepatic cells in vivo.

A study was made of the effects of certain inhibitors of transport systems on the actions of four cadmium (Cd) complexing N,N-disubstituted dithiocarbamates (DTCs) in mobilizing murine renal and hepatic Cd in vivo. Probenecid, the prototypical antagonist of organic anion transport in the kidney, when given 1 hr prior to each DTC, sharply suppressed the DTC-induced reduction of renal Cd but was virtually without effect on mobilization of Cd from liver. Sulfinpyrazone, which blocks tubular reabsorption of uric acid and also inhibits transport of a variety of organic acids, inhibited markedly the mobilization of both renal and hepatic Cd by DTCs. Phlorizin, an inhibitor of tubular sugar reabsorption, did not affect the Cd mobilizing actions of DTCs in any consistent fashion. We propose that the high degree of selectivity of DTCs in mobilizing renal and hepatic Cd is dependent, at least in part, upon active transport of DTCs into these tissues via the organic anion transport systems. This report presents the first evidence that compounds of the (R)2NCSS- class may gain access to intracellular space by an active, carrier-mediated process.     

Action of alpha-dendrotoxin on K+ currents in nerve terminal regions of axons in rat olfactory cortex.

1. In the rat olfactory cortex, unmyelinated axons give rise to synapses en passant. This tissue was used to study the pharmacology of axonal K(+)-currents. Responses were measured from a group of these axons as unclamped field currents, with a polarizable suction electrode. 2. A single stimulus to the axons elicited a tetrodotoxin-sensitive Na(+)-dependent transient K(+)-currents were revealed by positive polarization of the suction electrode and were manifest as a negative current following the Na(+)-component. 3. In the presence of tetraethylammonium (TEA, 5 mM) and Cd2+ (100 microM), the K(+)-component was depressed by 3,4-diaminopyridine (3,4-DAP; 1 to 20 microM; IC50 2.0 +/- 0.4 microM). alpha-Dendrotoxin (DTX; 15-1500 nM) also attenuated the aminopyridine-sensitive component (IC50 93 +/- 4 nM). At the highest DTX concentration, depression of the K(+)-current was incomplete, the residual K+ current being reduced by 3,4-DAP (0.1 to 5 microM). 4. These results indicate the presence of two aminopyridine-sensitive K+ currents in this preparation distinguished by their susceptibility to DTX.     

Bidirectional transport of NMDA receptor and ionophore in the vagus nerve

Accumulations of binding sites for [3H]3-[+-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic ([3H]CPP) and [3H]-N-(1-[2-thienyl]cyclohexyl)-3,4-piperidine ([3H]TCP) were demonstrated both proximal and distal to 24-h ligatures of the central and peripheral vagus nerve in the rat using radioligand binding and autoradiography. Peaks of label occurred at the proximal and distal extremities of double ligations but not between the ligatures, or in sham-operated or colchicine-treated nerves. Together these results suggest that both the primary acceptor site and the ionophore of the N-methyl-D-aspartate receptor undergo bidirectional axonal transport in central and peripheral branches of the vagus nerve.     

Accumulation of cadmium in human placenta interacts with the transport of micronutrients to the fetus

Cadmium (Cd) is a widespread, highly toxic environmental pollutant known to accumulate in human placenta. The aim of the present study was to elucidate to what extent the accumulation of Cd in human placenta interacts with the transport of micronutrients to the fetus. Cd and micronutrients were measured in placenta and umbilical cord blood from 44 non-smoking, rural Bangladeshi women, using ICPMS. Metallothionein (MT) protein expression was determined in placenta using Western blot. Cd in placenta (median 110 μg/kg dry weight, 20 μg/kg wet weight) was positively associated with maternal urinary Cd. It was also positively associated with Cd in umbilical cord blood (median 0.16 μg/kg), but negatively associated with zinc (Zn; median 3 mg/kg) in umbilical cord blood. Umbilical cord blood Zn was positively associated with birth anthropometry measures, and the Cd-related impairment of Zn in umbilical cord blood seemed to decrease size at birth. In multivariate analysis, MT protein expression was associated with Cd (positively) in placenta, but not with Zn or copper (Cu) in placenta. In conclusion, the Cd concentrations in placenta were clearly elevated, which seemed to impair Zn transfer to the fetus. Induction of MT explained the placental accumulation of Cd, but not the impairment of Zn transport.     

Role of the olfactory receptor neurons in the direct transport of inhaled uranium to the rat brain

Uranium presents numerous industrial and military uses and one of the most important risks of contamination is dust inhalation. In contrast to the other modes of contamination, the inhaled uranium has been proposed to enter the brain not only by the common route of all modes of exposure, the blood pathway, but also by a specific inhalation exposure route, the olfactory pathway. To test whether the inhaled uranium enter the brain directly from the nasal cavity, male Sprague–Dawley rats were exposed to both inhaled and intraperitoneally injected uranium using the ²³⁶U and ²³³U, respectively, as tracers. The results showed a specific frontal brain accumulation of the inhaled uranium which is not observed with the injected uranium. Furthermore, the inhaled uranium is higher than the injected uranium in the olfactory bulbs (OB) and tubercles, in the frontal cortex and in the hypothalamus. In contrast, the other cerebral areas (cortex, hippocampus, cerebellum and brain residue) did not show any preferential accumulation of inhaled or injected uranium. These results mean that inhaled uranium enters the brain via a direct transfer from the nasal turbinates to the OB in addition to the systemic pathway. The uranium transfer from the nasal turbinates to the OB is lower in animals showing a reduced level of olfactory receptor neurons (ORN) induced by an olfactory epithelium lesion prior to the uranium inhalation exposure. These results give prominence to a role of the ORN in the direct transfer of the uranium from the nasal cavity to the brain.     

Altered ovarian progesterone secretion induced by cadmium fails to interfere with embryo transport in the oviduct of the rat.

The effect of Cadmium (Cd) on embryo transport through the oviduct and on ovarian progesterone (P) secretion were studied in the rat. Animals were given 2.5, 5, 10 mg/kg CdCl2 or 1.0 mL/kg NaCl sc on day 1 of pregnancy. On days 1, 2, 3, 4, and 5, they were anesthetized with pentobarbital, cannulae were inserted in one of the utero-ovarian veins, and 5-minute blood samples were taken from the ovary. Ovarian venous outflow was recorded, P was determined from the blood fractions, and secretion rates were calculated. P levels were determined in peripheral blood. Body weights and the wet weight of adrenals, ovaries, and oviducts were checked; oviducts and uterine horns were flushed; and number, location, and developmental stage of embryos were observed. Cd content of the oviducts was measured. Cd accumulated dose and time dependently in oviducts and induced a dose-dependent depression and delay in the rise of ovarian P secretion during days 1 through 5 of pregnancy. In the peripheral blood, P levels also failed to rise until day 4 of pregnancy in Cd-treated rats. In embryo transfer, however, no alteration could be observed. It is hypothesized that lack of vascular contact in the oviduct makes it possible for the preimplantation embryos to escape toxic effects of Cd.     

Health effects of low-level cadmium intake and the role of metallothionein on cadmium transport from mother rats to fetus

Female Wistar rats were given Cd (as CdCl(2)) at a dose of 0, 1, 2, and 5 mgCd/kg/day by gastric tube daily for 6 consecutive days each week for 10 weeks. After the birth, newborn rats were sacrificed on day 1 and at 4 weeks. Mother rats were sacrificed after 4 weeks of lactation The concentrations of Cd in uterus and placenta, and metallothionein (MT) in the uterus of mother rats were determined. The concentrations of Cd in kidney and liver of newborn rats were also determined. Expression of iso-MT genes (I, II, and III) in the uterus of mother rats was measured using RT-PCR. The Cd concentration in the liver of newborn rats at the first day after birth was higher than in the kidney, while the concentration in the kidney of newborn rats at the fourth week after the birth was significantly higher than in the liver. The uterine MT concentration increased with accumulation of Cd; however, the MT concentration did not increase enough to prevent Cd transport to the fetus. On the other hand, it was considered that more Cd was transported as the chemical form of nonMT-Cd from mother rat, and accumulated in the liver rather than kidney of the fetus. Based on analyses of the Cd distribution in the liver and kidney of newborn rats, we speculate that MT in the uterus and placenta does not play a significant role in preventing Cd transport through the placenta from the uterus to the fetus.     

pH-Dependent transport of cadmium in rat renal brush border membrane vesicles: cadmium efflux via H+-antiport

The effects of pH on the Cd efflux across the renal brush border membrane were investigated using isolated rat renal brush border membrane vesicles (BBMV). BBMV equilibrated at pH 5.5 or 7.5 (pH_in) were mixed with an experimental buffer at pH 5.5 or 7.5 (pH_out) containing CdCl₂. After the incubation with CdCl₂, BBMV were washed with a chelating agent, EGTA, to remove Cd bound to the outer surface of BBMV. The initial accumulation of Cd in BBMV incubated for 1, 3 or 5 min at pH_in=5.5 and pH_out=7.5 (outwardly directed H⁺-gradient) was significantly higher than that at pH_in=pH_out=7.5, but the equilibrated Cd accumulation incubated for over 15 min was almost the same. FCCP, a protonophore, diminished the increasing effect of the H⁺-gradient on the initial Cd accumulation, whereas valinomycine, a K⁺ ionophore, did not. Kinetic analysis of the Cd accumulation incubated with different concentrations of CdCl₂ suggests that Cd is taken up into BBMV via the unsaturable component and saturable component and the outwardly directed H⁺-gradient increases the V_max value of the saturable component. The BBMV were incubated with CdCl₂ at different temperatures under the outwardly directed H⁺-gradient, and the increase in Cd accumulation due to the gradient was analyzed by an Arrhenius plot. The plot of increased Cd accumulation was nonlinear, suggesting the involvement of a carrier-mediated process. These findings suggest that the outwardly directed H⁺-gradient increases the Cd efflux across the renal brush border membrane via H⁺-antiport system.     

Carrier mediated transport of chlorpheniramine and chlorcyclizine across bovine olfactory mucosa: implications on nose-to-brain transport

Delivery to the CNS via the nasal cavity has been pursued as a means to circumvent the blood-brain barrier (BBB), yet the mechanism of drug transport across this novel route is not well understood. Hydroxyzine and triprolidine have been reported to readily reach the CNS following nasal administration, whereas no measurable amounts of chlorcyclizine or chlorpheniramine, structurally similar antihistamines, were observed in the CSF. The permeation of chlorpheniramine and chlorcyclizine in vitro across the bovine olfactory mucosa was studied to investigate the biological and physicochemical characteristics that contribute to the limited CNS disposition of these compounds following nasal administration. The submucosal to mucosal fluxes (J(s-m)) of chlorcyclizine and chlorpheniramine across the olfactory mucosa were significantly greater than the mucosal to submucosal fluxes (J(m-s)). Moreover, the submucosal-mucosal permeability of both compounds was temperature dependent and saturable. In the presence of metabolic inhibitors (ouabain and 2,4-dinitrophenol) and P-glycoprotein (P-gp)/multidrug resistance protein 1 (MRP1) inhibitors (quinidine and verapamil), the J(m-s) increased and J(s-m) decreased significantly. These results indicate that chlorpheniramine and chlorcyclizine are effluxed from the olfactory mucosa by efflux transporters such as P-gp and MRP1. Transport studies across inert polymeric membranes demonstrated that the permeability of chlorpheniramine and chlorcyclizine decreased at donor concentrations higher than 3 mM suggesting that physicochemical properties such as self-aggregation also play a role in the reduced olfactory mucosal permeability of these compounds at higher concentrations.