The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses.

In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury.

Differential gene expression of CYP3A isoforms in equine liver and intestines.

Recently, seven CYP3A isoforms - CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 - have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin-isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K(m) for the LIPA substrate in the liver and the intestine, while the maximum velocity (V(max)) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses.

Expression and localization of BCRP, MRP1 and MRP2 in intestines, liver and kidney in horse.

The gene and protein expression and the cellular localization of the ABC transport proteins breast cancer resistance protein (BCRP), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance-associated protein 2 (MRP2) have been examined in the intestines, liver and kidney in horse. High gene and protein expression of BCRP and MRP2 were found in the small intestines, with cellular localization in the apical membranes of the enterocytes. In the liver, MRP2 was present in the bile canalicular membranes of the hepatocytes, whereas BCRP was localized in the cytoplasm of hepatocytes in the peripheral parts of the liver lobuli. In the kidney both BCRP and MRP2 were predominantly present in the distal tubuli and in the loops of Henle. In most tissues, the gene and protein expression of MRP1 were much lower than for BCRP and MRP2. Immunostaining of MRP1 was detectable only in the intestines and with localization in the cytoplasm of enterocytes in the caecum and colon and in the cells of serous acini of Brunner's glands in the duodenum and the upper jejunum. The latter cells were also stained for BCRP, but not for MRP2. Many drugs used in horse are substrates for one or more of the ABC transport proteins. These transporters may therefore have important functions for oral bioavailability, distribution and excretion of substrate compounds in horse.

P-glycoprotein in intestines, liver, kidney and lymphocytes in horse.

P-glycoprotein (P-gp) is an important drug transporter, which is expressed in a variety of cells, such as the intestinal enterocytes, the hepatocytes, the renal tubular cells and the intestinal and peripheral blood lymphocytes. We have studied the localization and the gene and protein expression of P-gp in these cells in horse. In addition we have compared the protein sequence of P-gp in horse with the protein sequences of P-gp in several other species. Real time RT-PCR and Western blot showed gene and protein expression of horse P-gp in all parts of the intestines, but there was no strict correlation between these parameters. Immunohistochemistry showed localization of P-gp in the apical cell membranes of the enterocytes and, in addition, staining was observed in the intestinal intraepithelial and lamina propria lymphocytes. Peripheral blood lymphocytes also stained for P-gp, and gene and protein expression of P-gp were observed in these cells. There was a high gene and protein expression of P-gp in the liver, with P-gp-immunoreactivity in the bile canalicular membranes of the hepatocytes. Gene and protein expression of P-gp were found in the kidney with localization of the protein in different parts of the nephrons. Protein sequence alignment showed that horse P-gp has two amino acid insertions at the N-terminal region of the protein, which are not present in several other species examined. One of these is a 99 amino acid long sequence inserted at amino acid positions 23-121 from the N-terminal. The other is a six amino acid long sequence present at the amino acid positions 140-145 from the N-terminal. The results of the present study indicate that P-gp has an important function for oral bioavailability, distribution and excretion of substrate compounds in horse.

Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse.

Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b(5) and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b(5) in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b(5).

Clinical signs and etiology of adverse reactions to procaine benzylpenicillin and sodium/potassium benzylpenicillin in horses.

Case reports of 59 horses reacting adversely to procaine benzylpenicillin or to sodium or potassium benzylpenicillin in Sweden in 2003-2005 were obtained through contacts with horse-owners. For the assessment of the reports, various parameters were evaluated, such as the times to the reactions, information on previous penicillin treatment, the clinical signs and the actions taken in the reacting horses. Among the reports, two horses had received sodium or potassium benzylpenicillin intravenously, whereas the remaining 57 horses had been treated with procaine benzylpenicillin intramuscularly. Allergy may underlie the adverse reactions in the horses given sodium and potassium benzylpenicillin, and in a few of the horses given procaine benzylpenicillin. However, in most horses in the latter group, the clinical signs may be due to the toxic effects of procaine. In these horses, the dominating clinical signs were locomotor and behavioral changes. Some risk factors may enhance the probability that horses react to procaine. One is repeated injections, which increase the likelihood of intravascular administration and also may increase the sensitivity to procaine due to neuronal sensitization (kindling). Procaine is rapidly hydrolyzed by plasma esterases to nontoxic metabolites. When high amounts of procaine enter the circulation, the hydrolyzing capacity may be exceeded and toxicity occurs. Analyses of plasma esterases from reacting horses showed lower activity than in nonreacting control horses. Low esterase activity may increase the possibility of procaine toxicity and constitute another risk factor.

Cetirizine in horses: pharmacokinetics and effect of ivermectin pretreatment.

The pharmacokinetics of the histamine H(1)-antagonist cetirizine and the effects of pretreatment with the antiparasitic macrocyclic lactone ivermectin on the pharmacokinetics of cetirizine were studied in horses. After oral administration of cetirizine at 0.2 mg/kg bw, the mean terminal half-life was 3.4 h (range 2.9-3.7 h) and the maximal plasma concentration 132 ng/mL (101-196 ng/mL). The time to reach maximal plasma concentration was 0.7 h (0.5-0.8 h). Ivermectin (0.2 mg/kg bw) given orally 1.5 h before cetirizine did not affect its pharmacokinetics. However, ivermectin pretreatment 12 h before cetirizine increased the area under the plasma concentration-time curve by 60%. The maximal plasma concentration, terminal half-life and mean residence time also increased significantly following the 12 h pretreatment. Ivermectin is an inhibitor of P-glycoprotein, which is a major drug efflux transporter in cellular membranes at various sites. The elevated plasma levels of cetirizine following the pretreatment with ivermectin may mainly be due to decreased renal secretion, related to inhibition of the P-glycoprotein in the proximal tubular cells of the kidney. The pharmacokinetic properties of cetirizine have characteristics which are suitable for an antihistamine, and this substance may be a useful drug in horses.

Fexofenadine in horses: pharmacokinetics, pharmacodynamics and effect of ivermectin pretreatment.

The pharmacokinetics and the effects on inhibition of histamine-induced cutaneous wheal formation of the histamine H1-antagonist fexofenadine were studied in horse. The effect of ivermectin pretreatment on the pharmacokinetics of fexofenadine was also examined. After intravenous infusion of fexofenadine at 0.7 mg/kg bw the mean terminal half-life was 2.4 h (range: 2.0-2.7 h), the apparent volume of distribution 0.8 L/kg (0.5-0.9 L/kg), and the total body clearance 0.8 L/h/kg (0.6-1.2 L/h/kg). After oral administration of fexofenadine at 10 mg/kg bw bioavailability was 2.6% (1.9-2.9%). Ivermectin pretreatment (0.2 mg/kg, p.o.) 12 h before oral fexofenadine decreased the bioavailability to 1.5% (1.4-2.1%). In addition, the area under the plasma concentration-time curve decreased 27%. Ivermectin did not affect the pharmacokinetics of i.v. administered fexofenadine. Ivermectin may influence fexofenadine absorption by interfering in intestinal efflux and influx pumps, such as P-glycoprotein and the organic anion transport polypeptide family. Oral and i.v. fexofenadine significantly decreased histamine-induced wheal formation, with a maximal duration of 6 h. A pharmacokinetic/pharmacodynamic link model indicated that fexofenadine in horse has antihistaminic effects at low plasma concentrations (EC50 = 16 ng/mL). However, oral treatments of horses with fexofenadine may not be suitable due to the low bioavailability.

Cell-specific activation of aflatoxin B1 correlates with presence of some cytochrome P450 enzymes in olfactory and respiratory tissues in horse.

Horses may be exposed to aflatoxin B(1) (AFB(1)) via inhalation of mouldy dust, leading to high exposure of olfactory and respiratory tissues. In the present study the metabolic activation of AFB(1) was examined in olfactory and respiratory tissues in horse. The results showed covalent binding of AFB(1)-metabolites in sustentacular cells and cells of Bowman's glands in the olfactory mucosa, in some cells of the surface epithelium of nasal respiratory, tracheal, bronchial and bronchiolar mucosa and in some glands in these areas. Immunohistochemistry revealed that cells expressing proteins reacting with CYP 3A4- and CYP 2A6/2B6-antibodies had a similar distribution as those having capacity to activate AFB(1). Our data indicate that the cell-specific activation of AFB(1) correlates with presence of some CYP-enzymes in olfactory and respiratory tissues in horse.

Fine-scale tissue distribution of cadmium, inorganic mercury, and methylmercury in nymphs of the burrowing mayfly Hexagenia rigida studied by whole-body autoradiography.

The distribution of inorganic 109Cd(II), inorganic 203Hg(II), and [203Hg] methylmercury (MeHg) in nymphs of the burrowing mayfly Hexagenia rigida after exposure via water and sediments was studied. To better understand the mechanisms underlying the fate of Cd, Hg, and MeHg in this animal and to identify target organs, autoradiography of whole-body cryosections was used to obtain a detailed view of the distribution of the radiolabels. The gut and exoskeleton were the only structures labeled in nymphs exposed to Cd via water or sediments. After exposure to inorganic Hg via water, the Malpighian tubules exhibited a very high labeling, indicating that these organs may be a target for Hg toxicity. The distribution of Hg after exposure via sediments was similar, though the labeling of Malpighian tubules was less intense. Distribution of MeHg strongly differed between treatment groups. Nymphs were rather uniformly labeled after exposure via water, whereas in those exposed to MeHg in sediments, the intense labeling of all internal tissues contrasted with the very low labeling of the hemolymph, indicating that the translocation rate of the absorbed MeHg was faster in the latter group. This may be related to the complexation of MeHg by small thiol ligands in the gut as a result of the digestion process.

Cytochrome P450 expression and related metabolism in human buccal mucosa.

Constituents in food and fluids, tobacco chemicals and many drugs are candidates for oral absorption and oxidative metabolism. On this basis, the expression of cytochrome P450 isozymes (CYPs) and the conversion of CYP substrates were analysed in reference to buccal mucosa. A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5. CYP 2D6 was expressed in six out of the 13 specimens, whereas all samples were negative for 2A6 and 2B6. Serum-free monolayer cultures of the Siman virus 40 large T-antigen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte lines expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1). Dealkylation of ethoxyresorufin and methoxyresorufin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for both substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1. Absent or minute catalytic activity of 2C9, 2D6 and 3A4 in the various cell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2-0.5 pmol/min/mg). Metabolic activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin aflatoxin B1 (AFB1) to covalently bound adducts was indicated by autoradiographic analysis of both monolayer and organotypic cultures of SVpgC2a. In contrast, SqCC/Y1 showed lower or absent metabolic activity for these substrates. Finally, measurements of various non-reactive AFB1 metabolites indicated rates of formation <0.1 pmol/min/mg in both normal and transformed cells. The results indicate presence of several CYPs of which some may contribute to significant xenobiotic metabolism in human buccal epithelium. Notably, metabolic activation of AFB1 was not previously implicated for oral mucosa. Further, the results show that CYP-dependent metabolism can be preserved or even activated in immortalized keratinocytes. Metabolic activity in SVpgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies.

Intranasal instillation of aflatoxin B(1) in rats: bioactivation in the nasal mucosa and neuronal transport to the olfactory bulb.

Aflatoxin B(1) (AFB(1)) may be present in moldy dust. Inhalation of contaminated dust particles may result in high local exposure of the nasal mucosa. The present study was designed to assess bioactivation and toxicity of AFB(1) in the nasal mucosa after intranasal administration of the mycotoxin in rats and also to examine if translocation of the mycotoxin occurs from the nasal mucosa to the brain along olfactory neurons. Female Sprague-Dawley rats were given (3)H-AFB(1) (0.2, 1 or 20 microg) intranasally and were sacrificed at various intervals (1 h to 20 d). Tissues were examined autoradiographically or histopathologically. Quantitative data were obtained by beta-spectrometry in rats given (3)H-AFB(1) intranasally or orally (for comparison). The data indicated that intranasal administration of AFB(1) resulted in formation of tissue-bound metabolites in sustentacular cells, in some cells of Bowman's glands, and in a population of neuronal cells in the olfactory mucosa, whereas in the respiratory nasal mucosa, there was selective bioactivation of AFB(1) in mucous cells. Intranasal instillation of 20 microg AFB(1) resulted in disorganized undulating olfactory epithelium, with injured neuronal and sustentacular cells. In the respiratory epithelium, there was selective destruction of mucous cells. beta-Spectrometry and autoradiography with tape-sections of the head of rats given (3)H-AFB(1) intranasally indicated transport of AFB(1) and/or AFB(1) metabolites along the axons of the primary olfactory neurons to their terminations in the glomeruli of the olfactory bulb. The data indicate that the materials transported in the olfactory nerves represent AFB(1) and/or some of its nonreactive metabolites. It is concluded that application of AFB(1) on the nasal mucosa in rats results in high local bioactivation of the mycotoxin in this tissue and translocation of AFB(1) and/or its metabolites to the olfactory bulb.

Manganese taken up into the CNS via the olfactory pathway in rats affects astrocytes.

Manganese (Mn), administered intranasally in rats, is effectively taken up in the CNS via the olfactory system. In the present study, Mn (as MnCl(2)) dissolved in physiological saline, was instilled intranasally in rats at doses of 0 (control), 10, 250, or 1000 microg. At the start of the experiment each rat received an intranasal instillation. Some rats were killed after one week without further treatment (the 1-w group), whereas the remaining rats received further instillations after one and two weeks and were killed after an additional week (the 3-w group). The brains were removed and either used for ELISA-determination of the astrocytic proteins glial fibrillary acidic protein (GFAP) and S-100b or histochemical staining of GFAP and S-100b, microglia (using an antibody against the iba1-protein) and the neuronal marker Fluoro-Jade. There were no indications that the Mn induced neuronal damage. On the other hand, the ELISA showed that both GFAP and S-100b decreased in the olfactory cortex, the hypothalamus, the thalamus, and the hippocampus of the 3-w group. The only effect observed in the 1-w group was a decrease of S-100b in the olfactory cortex at the highest dose. The immunohistochemistry showed no noticeable reduction in the number of astrocytes. We assume that the decreased levels of GFAP and S-100b are due to an adverse effect of Mn on the astrocytes, although this effect does not result in astrocytic demise. In the 3-w group, exposed to the highest dose of Mn, increased levels of GFAP and S-100b were observed in the olfactory bulbs, but these effects are probably secondary to a Mn-induced damage of the olfactory epithelium. Our results indicate that the astrocytes are the initial targets of Mn toxicity in the CNS.

Uptake of metals in the brain via olfactory pathways.

In the olfactory epithelium the dendrites of the primary olfactory neurons are in contact with the nasal lumen, and via the axons these neurons are also connected to the olfactory bulbs of the brain. Materials which come into contact with the olfactory epithelium can be taken up in the primary olfactory neurons and be transported to the olfactory bulbs and even further into other areas of the brain. The present review deals with the mechanism of uptake and transport of metals in the olfactory system. Metals discussed are mainly manganese, cadmium, nickel and mercury. Among the metals so far examined, manganese has been found to have a unique capacity to be taken up via the olfactory pathways and pass transneuronally to other parts of the brain. It is considered that the occupational neurotoxicity of inhaled manganese may be related to an uptake of the metal into the brain via the olfactory pathways. Studies with nickel indicate that this metal, following a transport to the terminal parts of the primary olfactory neurons in the glomeruli of the bulbs, slowly passes to secondary and tertiary olfactory neurons. Cadmium and mercury are transported along the primary olfactory neurons to their terminations in the olfactory bulbs, but these metals appear unable to continue along secondary olfactory neurons. Occupational inhalation of nickel or cadmium can be toxic to the olfactory sense. It is not yet known whether mercury is toxic to the olfactory system in mammals, but this metal is known to alter olfaction and olfactory-related behaviour in fish. Data in the literature dealing with a potential olfactory-related neurotoxicity of aluminum are also discussed in the paper.

Transport of manganese via the olfactory pathway in rats: dosage dependency of the uptake and subcellular distribution of the metal in the olfactory epithelium and the brain.

The dosage dependency of the uptake of Mn from the olfactory epithelium via olfactory neurons into the brain was studied after intranasal administration of the metal in rats. The results indicate that the Mn transport is saturable both regarding the uptake into the olfactory epithelium and the transfer to the olfactory bulb. Further, our data indicate that Mn moves relatively freely from the olfactory bulb to the olfactory cortex at an amount dependent on the level of influx into the bulb. The transport to the rest of the brain was related to the amounts in the olfactory bulb and the olfactory cortex, but the relative proportion reaching this area increased with increasing doses. Cell fractionations showed that the Mn was present both in the cytosol and in association with various cell constituents. Gel filtrations of the cytosol on a Superdex 30 column showed that about 20% of the Mn in the brain and about 3% in the olfactory epithelium was eluted together with high-molecular-weight materials (MW > 10,000), whereas the rest was eluted in the total volume and may represent unbound metal. It is likely that the metal has been loosely associated with protein(s) or other constituents at the application to the column, but that this association is too loose to be retained during the passage through the column. Our results show that the olfactory neurons provide a pathway with a considerable capacity to transport Mn into the brain. We propose that the neurotoxicity of inhaled Mn is related to an uptake via this route.

Transport of nickel across monolayers of human intestinal Caco-2 cells.

The passage of nickel across monolayers of intestinal epithelial Caco-2 cells, originally derived from a human colonic adenocarcinoma, was studied in bicameral chambers. The results showed that the transport and accumulation of nickel were depressed in iron-loaded monolayers, indicating that the metal participates in an absorptive process for iron in the Caco-2 cells. No detectable transport of nickel in either the apical to basal or basal to apical direction occurred at 4 degreesC. Since cellular metabolism is inhibited at 4 degreesC, these data indicate that there is no passive transcellular or paracellular passage of the nickel across the monolayers. Studies in ATP-depleted monolayers showed an increased permeability of nickel, and concomitantly there was a similar increase in the permeability of the paracellular marker mannitol. These results indicate that the metabolic inhibition results in a loosening of the junctional complexes between the Caco-2 cells, resulting in a paracellular leakage of the nickel. Additional experiments showed that the transport of nickel in the basal to apical direction occurred at a higher rate than in the apical to basal direction. This indicates the presence of an extrusion mechanism that secretes the nickel from the basal to the apical side of the Caco-2 cells. Studies with Caco-2 cells and in vivo studies by other authors have shown similar results for other metals, indicating that colonic epithelial cells may have the ability to secrete some metals.

Transport and subcellular distribution of nickel in the olfactory system of pikes and rats.

Occupational exposure to nickel by inhalation may result in impaired olfactory sense. Recent studies have shown that nickel is transported from the olfactory epithelium along the axons of the primary olfactory neurons to the brain. In the present study 63Ni2+ was applied in the olfactory chambers of pikes (Esox lucius) and the rate at which the metal was transported in the primary olfactory neurons was determined by beta-spectrometry. The results showed a wave of 63Ni2+ in the olfactory nerves, which slowly moved toward the olfactory bulbs. The maximal 63Ni2+ transport rate corresponding to the movement of the base of the wave front was found to be about 0.13 mm/h at the experimental temperature (10 degrees C). This rate of 63Ni2+ transport falls into the class of slow axonal transport. Radioluminography of tape sections of a pike given 63Ni2+ in the right olfactory chamber showed a selective labeling of the right olfactory nerve. The subcellular distribution of 63Ni2+ in the olfactory nerves and the olfactory epithelium of the pikes was studied in tissues subjected to homogenizations and centrifugations, and these methods were also used to examine the subcellular distribution of 63Ni2+ in tissues of the olfactory system of rats given the metal intranasally. It was found that the 63Ni2+, in both the pike and the rat, was present in the cytosol and also in association with various particulate cell constituents. Gel filtrations of the cytosols showed that the 63Ni2+ mainly was eluted at a Ve/Vo ratio corresponding to a MW of about 250. The same coefficient was obtained in gel filtrations performed with 63Ni2+ mixed with histidine in vitro. It is likely that the cytosolic nickel may be bound to histidine or possibly to other amino acids which are similar in size to histidine. Additionally, in the olfactory tissues of the rat the 63Ni2+ was partly present in the cytosol in association with a component with a MW of about 25,000. It is concluded that (i) 63Ni2+ is transported in the primary olfactory neurons by means of slow axonal transport, (ii) in this process the metal is bound to both particulate and soluble cytosolic constituents, and (iii) the metal shows this subcellular distribution also in other parts of the olfactory system.

Uptake of inorganic mercury in the olfactory bulbs via olfactory pathways in rats.

Uptake and transport in the olfactory neurons may be an important means by which some heavy metals gain access to the brain. In the present study we explored whether inorganic mercury (203Hg2+) may be taken up in the CNS via the olfactory pathway. Autoradiography and gamma spectrometry showed that intranasal instillation of 203Hg2+ in the right nostrils of rats resulted in much higher levels of the metal in the right olfactory bulbs than in the left ones. At the side of the application of the 203Hg2+ there was also a labeling of the olfactory nerve bundles projecting to the olfactory bulbs as well as in the olfactory nerve-fibres constituting the olfactory nerve layer of the bulbs, which was not seen on the opposite side. The results also showed that the 203Hg2+ accumulated in the glomerular layer of the bulbs. These data indicate that our results can be ascribed to a movement of the mercury along the olfactory axons to their terminal parts in the glomeruli and not to circulatory uptake from the mucosal vasculature. At late survival intervals a low labeling was also discernable in the external plexiform layer, indicating that a low level of 203Hg2+ leaves the terminal arborizations of the axons in the glomeruli. An uptake of 203Hg2+ in the glomerular layer of the olfactory bulbs was also seen in rats given the metal intraperitoneally. This uptake was similar in the right and left bulbs and always much lower than in the right bulbs of the rats given 203Hg2+ in the right nostrils. The intraperitoneal injections in addition resulted in an uptake of the 203Hg2+ in the olfactory epithelium. We propose that in these rats the mercury is taken up from the blood into the olfactory neurons and then moves along the axons to their terminations in the olfactory bulbs. In humans a continuous exposure of the nasal cavity to mercury vapor (Hg0), released from amalgam fillings and oxidized to Hg2+ in the olfactory mucosa, as well as a potential uptake of Hg2+ in the olfactory neurons from the blood, may lead to considerable concentrations of the metal in the olfactory bulbs.

Binding of 3H-metronidazole in olfactory, respiratory and alimentary epithelia in rats.

Whole-body autoradiography of 3H-metronidazole in rats showed retention of bound metabolites in the epithelia lining the olfactory part of the nose, the tongue, the gingiva, the palate, the pharynx, the oesophagus and the forestomach. In vitro microautoradiography in O2- and N2-atmosphere with some of these tissues indicated reductive formation of bound metabolites in specific cells of the epithelia. Studies with subcellular fractions of the nasal olfactory mucosa showed formation of DNA- and protein-bound metronidazole metabolites. A lower bioactivating capacity was found in experiments with the liver. The bioactivation was dependent on N2-atmosphere, and presence of the P450-inhibitor metyrapone or GSH in the incubation media depressed the protein-binding of metronidazole both in the nasal olfactory mucosa and the liver. These data indicate that the bioactivation is partly P450-dependent and GSH may play an important role in scavaging the bioactivated drug. The epithelial cells with a capacity to bioactivate metronidazole may be potential targets for negative effects of the drug. Whole-body autoradiography also showed a strong binding of radioactivity in the contents of caecum and colon. This can be considered to be due to reductive bioactivation of metronidazole by the intestinal microorganisms and reflects the principal site of action of the drug.

Effect of dietary iron-deficiency on the disposition of nickel in rats.

Nickel was given orally to iron-deficient and iron-sufficient rats and the levels of the metal in various tissues were examined at several time intervals. The results showed higher levels of nickel in the tissues of the iron-deficient rats, as compared to the iron-sufficient ones, 3, 6, 24, 48 and 120 h following gastric intubation of the metal. The results also showed higher levels of the metal in some tissues of iron-deficient rats than in iron-sufficient ones 24 h after intra-peritoneal nickel administration. A lower urinary excretion of nickel was observed in the iron-deficient rats given the intraperitoneal injections, as compared to the iron-sufficient animals. Our results indicate that nickel, at least in part, is taken up by the absorptive mechanism for iron in the intestinal epithelium. In addition, the iron-status appears to affect the uptake of nickel from the blood into the tissues.