Evaluation of the toxicity of zinc in the rat olfactory neuronal cell line, Odora.

Zinc (Zn) has long been touted as a panacea for common cold. Recently, there has been some controversy over whether an intranasal (IN) zinc gluconate gel, purported to fight colds, causes anosmia, or loss of the sense of smell, in humans. Previous evidence has shown that IN zinc sulfate (ZnSO4) solutions can cause anosmia in humans as well as significant damage to the olfactory epithelium in rodents. Using an in vitro olfactory neuron model (the rat Odora cell line), we tested the hypothesis that Zn toxicity was caused by inhibition of the hydrogen voltage-gated channel 1(HVCN1), leading to acidosis and apoptotic cell death. Following studies to characterize the toxicity of zinc gluconate and ZnSO4, Odora cells were grown on coverslips and loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH in the presence and absence of Zn salts. While we found that HVCN1 is not functional in Odora cells, we found that olfactory neurons in vitro maintain their intracellular pH through a sodium/proton exchanger, specifically the sodium proton antiporter 1. ZnSO4, at nontoxic levels, had no impact on intracellular pH after acute exposure or after 24 h of incubation with the cells. In conclusion, Zn toxicity is not mediated through an acidification of intracellular pH in olfactory neurons in vitro.

Protection from olanzapine-induced metabolic toxicity in mice by acetaminophen and tetrahydroindenoindole.

OBJECTIVE: In mice and in humans, treatment with the second-generation antipsychotic drug olanzapine (OLZ) produces excessive weight gain, adiposity and secondary metabolic complications, including loss of glucose and insulin homeostasis. In mice consuming a high-fat (HF) diet, a similar phenotype develops, which is inhibited by the analgesic acetaminophen (APAP) and by the antioxidant tetrahydroindenoindole (THII). Therefore, we examined the ability of APAP and THII to prevent metabolic changes in mice receiving OLZ. DESIGN AND MEASUREMENT: C57BL/6J mice received either a normal diet or a HF diet, and were administered daily dosages of OLZ (3 mg kg(-1) body weight), alone or with APAP (30 mg kg(-1) body weight) or THII (4.5 mg kg(-1) body weight), for 10 weeks. Parameters of body composition and metabolism, including glucose and insulin homeostasis and oxidative stress, were examined. RESULTS: OLZ treatment doubled the HF diet-induced increases in body weight and percent body fat. These increases were partially prevented by both APAP and THII, although food consumption was constant in all groups. The THII protection was associated with an increase in whole body and mitochondrial respiration. OLZ also exacerbated, and both APAP and THII prevented, HF diet-induced loss of glucose tolerance and insulin resistance. As increased body fat promotes insulin resistance by a pathway involving oxidative stress, we evaluated production of reactive oxygen and lipid peroxidation in white adipose tissue (WAT). HF diet caused an increase in lipid peroxidation, NADPH-dependent O(2) uptake and H(2)O(2) production, which were further exacerbated by OLZ. APAP, THII and the NADPH oxidase inhibitor, diphenyleneiodonium chloride, each abolished oxidative stress in WAT. CONCLUSIONS: We conclude that both APAP and THII intervene in the development of obesity and metabolic complications associated with OLZ treatment.

Comparison of rat olfactory mucosal responses to carcinogenic and non-carcinogenic chloracetanilides.

Alachlor and butachlor are chloracetanilide herbicides that induce olfactory tumors in rats, whereas propachlor does not. The mechanism by which alachlor induces tumors is distinct from many other nasal carcinogens, in that alachlor induces a gradual de-differentiation of the olfactory mucosa (OM) to a more respiratory-like epithelium, in contrast to other agents that induce cytotoxicity, followed by an aberrant regenerative response. We studied biochemical and genomic effects of these compounds to identify processes that occur in common between alachlor- and butachlor-treated rats. Because we have previously shown that matrix metalloproteinase-2 (MMP2) is activated in OM by alachlor, in the present studies we evaluated both MMP2 activation and changes in OM gene expression in response to carcinogenic and non-carcinogenic chloracetanilide treatments. All three chloracetanilides activated MMP2, and >300 genes were significantly up- or downregulated between control and alachlor-treated rats. The most significantly regulated gene was vomeromodulin, which was dramatically upregulated by alachlor and butachlor treatment (>60-fold), but not by propachlor treatment. Except for similar gene responses in alachlor- and butachlor-treated rats, we did not identify clear-cut differences that would predict OM carcinogenicity in this study.

Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(-/-) knockout mice having increased hepatic BaP-DNA adduct levels.

Previous studies have shown that cytochrome P450 1A1 (CYP1A1), CYP1B1, and prostaglandin-endoperoxide synthase (PTGS2) are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), and all three metabolize BaP to reactive DNA-binding intermediates and excreted products. Because these three enzymes show differing patterns of basal levels, inducibility, and tissue-specific expression, animal studies are necessary to delineate the role of CYP1A1 in BaP-mediated toxicity. In mice receiving large daily doses of BaP (500 mg/kg i.p.), Cyp1a1(-/-) knockout mice are protected by surviving longer than Cyp1a1(+/-) heterozygotes. We found that a single 500 mg/kg dose of BaP induces hepatic CYP1A1 mRNA, protein, and enzyme activity in Cyp1a1(+/-) but not in Cyp1a1(-/-) mice; TCDD pretreatment increases further the CYP1A1 in Cyp1a1(+/-) but not Cyp1a1(-/-) mice. Although a single 500 mg/kg dose of BaP was toxic to Cyp1a1(+/-) mice (serum liver enzyme elevated about 2-fold above control levels at 48 h), Cyp1a1(-/-) mice displayed no hepatotoxicity. Unexpectedly, we found 4-fold higher BaP-DNA adduct levels in Cyp1a1(-/-) than in Cyp1a1(+/-) mice; TCDD pretreatment lowered the levels of BaP-DNA adducts in both genotypes, suggesting the involvement of other TCDD-inducible detoxification enzymes. BaP was cleared from the blood much faster in Cyp1a1(+/-) than Cyp1a1(-/-) mice. Our results suggest that absence of the CYP1A1 enzyme protects the intact animal from BaP-mediated liver toxicity and death, by decreasing the formation of large amounts of toxic metabolites, whereas much slower metabolic clearance of BaP in Cyp1a1(-/-) mice leads to greater formation of BaP-DNA adducts.

Increased activity of CYP3A enzyme in primary cultures of rat hepatocytes treated with docetaxel: comparative evaluation with paclitaxel.

PURPOSE: Docetaxel, a potent antimicrotubule agent widely used in the treatment of ovarian, breast and lung cancer, is extensively metabolized in various animal species, including humans. The metabolism of docetaxel to its primary metabolite, hydroxydocetaxel, is mediated by cytochrome P450 isozymes CYP3A2 and CYP3A4 in rats and humans, respectively. Several substrates of enzymes belonging to the CYP3A subfamily are known to induce different CYP isozymes, including CYP3A enzymes. Recently, paclitaxel, a compound structurally related to docetaxel, has been shown to significantly elevate the expression of CYP3A in rat and human hepatocytes. In this study we investigated the influence of docetaxel, employed at clinically relevant concentrations, on the level and the activity of cytochrome P450 3A in primary cultures of rat hepatocytes. METHODS: Rat hepatocytes were treated with different concentrations of docetaxel, paclitaxel and other CYP3A inducers. Testosterone 6beta-hydroxylase activity of intact hepatocytes was used as a marker for CYP3A. The immunoreactive CYP3A levels in the S-9 fractions were determined by Western blot analysis. RESULTS: We observed that by day 3 of drug treatment, docetaxel at concentration in the range of 2.5-10 microM increased the CYP3A enzymatic activity and the immunoreactive CYP3A levels in a concentration-dependent manner. At the 10 microM level, docetaxel caused a twofold increase in the CYP3A activity and a threefold increase in the immunoreactive CYP3A levels. However, the docetaxel-mediated CYP3A activity and enzyme level increase were significantly lower than those mediated by paclitaxel and dexamethasone. A comparison of the testosterone 6beta-hydroxylation activity in hepatocytes treated with these agents at a concentration of 5 microM each yielded the following rank order of induction capacity: dexamethasone > paclitaxel > docetaxel (15-fold, 5-fold, 2.2-fold, respectively). CONCLUSIONS: Taken together, our findings raise the possibility that docetaxel at clinically relevant concentrations increases CYP3A activity. The potential for docetaxel-mediated changes in the metabolism of other coadministered drugs and its own metabolism, in relation to that due to paclitaxel, are discussed.

Evolution of alachlor-induced nasal neoplasms in the Long-Evans rat.

The chloracetanilide herbicide alachlor (2-chloro-2',6-diethyl-N-(methoxymethyl)-acetanilide) induces nasal neoplasms in rats following chronic dietary exposure. The present study sought to identify the cellular origin and mechanisms of tumor induction and progression. Male Long-Evans rats were fed alachlor (0 or 126 mg/kg/day) beginning at 6 weeks of age. Following 1 month of alachlor ingestion, neither histological abnormalities nor enhanced cell division (assessed by BrdU incorporation) occurred in any region of the nasal cavity. Six months of alachlor exposure resulted in proliferation of basal and nonbasal cells in the olfactory mucosa while inducing nasal masses in 7 of 15 animals. Tumors ranged from dysplastic plaques to polypoid adenomas and originated in the olfactory regions of the nasal cavity. Neoplasms were associated with regions of respiratory metaplasia and were often covered with a low cuboidal, poorly ciliated epithelium. Tumor cells did not express characteristics of the olfactory mucosa, including olfactory marker protein (OMP, for neurons) and NMa (antibody recognizing cytochrome P450 [CYP] 2A3, found in Bowman's glands). Sites of plaque and tumor development coincided with regions of NMa immunoreactivity. These data suggest that local metabolism is important in alachlor-induced olfactory tumors and support the concept that metaplastic respiratory epithelial cells give rise to the observed neoplasms.

Targeted knockout of Cyp1a1 gene does not alter hepatic constitutive expression of other genes in the mouse [Ah] battery.

Using the Cre-lox system, we have generated a cytochrome P450 1A1 Cyp1a1(-/-) knockout mouse by deletion of the translated portions of the Cyp1a1 gene. These mice are viable and demonstrate no obvious phenotype, compared with wild-type littermates. As a first step toward characterizing genes that might be expected to compensate for loss of CYP1A1, constitutive expression of [Ah] gene battery members was examined. In a cultured hepatoma CYP1A1 metabolism-deficient mutant line that does not express Cyp1a2, we have previously shown that constitutive transcriptional up-regulation of other [Ah] gene battery members occurs; these results are consistent with the elevation of a putative endogenous ligand (EL) for the Ah receptor that is a substrate for CYP1A1. The [Ah] battery includes Cyp1a2, NAD(P)H:quinone oxidoreductase (Nqo1), and three other Phase II genes. Examining mRNA, protein, and enzyme activity, we demonstrate that the absence of CYP1A1 has no effect on the hepatic constitutive expression of Cyp1a2 or Nqo1. We postulate that CYP1A1 and CYP1A2 might have overlapping substrate specificity for metabolism of the EL, such that basal CYP1A2 in the liver can compensate for the loss of CYP1A1.

Characterization of lidocaine metabolism by rat nasal microsomes: implications for nasal drug delivery.

Lidocaine has been recently approved for use as an intranasal spray in the treatment of migraine. In this study, we investigated the metabolism of lidocaine to its primary metabolite monoethylglycine xylidide (MEGX), by rat nasal olfactory and respiratory microsomes. The metabolic parameters were compared with metabolism employing rat and human hepatic microsomes. The olfactory and respiratory microsomes both exhibited considerable activity for conversion of lidocaine to MEGX in comparison with the activity in the hepatic tissues. The rat olfactory microsomes had a markedly higher affinity than the rat hepatic or respiratory microsomes. However, the turnover rate was only about one-half that of rat liver. Employing Western immunoblotting we investigated the presence of cytochrome P450s (CYPs) 1A2, 3A2, 2B1 and 2C11 in rat nasal tissues; these isozymes are known to partcipated in the metabolism of lidocaine in rat liver. These isozymes were found to be present in significant amounts in both the nasal olfactory and respiratory tissue; this is the first known report of the presence of CYP2C11 in nasal mucosae. Our studies underscore the importance of CYP-mediated drug metabolism in nasal tissues. The effect of this 'nasal first-pass' should be weighed carefully while considering the fate and the bioavailability of drugs delivered via the intranasal route.

Evidence for site-specific bioactivation of alachlor in the olfactory mucosa of the Long-Evans rat.

Alachlor (2-chloro-2',6'-diethyl-N-[methoxymethyl]-acetanilide) is a restricted-use chloracetanilide herbicide which has been shown previously to produce a dose-dependent incidence of olfactory mucosal tumors in rats following chronic dietary exposure. However, the mechanism of alachlor carcinogenicity is poorly understood. Alachlor was administered i.p. to male Long-Evans rats for up to 28 days at doses that are carcinogenic in chronic studies in order to study olfactory lesion development and alterations in cell proliferation. Neither treatment-related olfactory mucosal lesions nor regenerative cell proliferation, as assessed with BrdU labeling, was detected. In vitro genotoxicity studies using Salmonella typhimurium strain TA100 showed that alachlor was non-mutagenic in the absence of metabolic activation. When pre-incubated with an olfactory mucosal S9 activation system, alachlor induced a weak, dose-dependent mutagenic response at 500-1250 micrograms/plate, with toxicity at higher doses. In contrast, an S9 activation system derived from nasal respiratory mucosa, the tissue physically juxtaposed with the olfactory mucosa but reportedly not susceptible to alachlor-induced tumors, did not produce a mutagenic response for alachlor or the positive control. Thus, this result suggested site-specificity of alachlor activation consistent with the target site of carcinogenicity. The mutagenicity of alachlor to Salmonella, in the presence of an olfactory mucosal-activating system, was confirmed by a limited positive response in the mouse lymphoma assay. Here there were increases in small colony mutants (indicative of chromosomal effects) as well as large colony mutants (which reflect gene mutations). This study suggests that target tissue bioactivation of alachlor results in the formation of one or more mutagenic metabolite(s), which may be critical in alachlor-induced nasal tumorigenesis.

Role of CYP2A5 and 2G1 in acetaminophen metabolism and toxicity in the olfactory mucosa of the Cyp1a2(-/-) mouse.

Acetaminophen (AP) is a widely-used analgesic agent that has been linked to human liver and kidney disease with prolonged or high-dose usage. In rodents, the target organs that are affected include liver, kidney, and the olfactory mucosa. AP toxicity requires cytochrome P450(CYP)-mediated metabolic activation, and the isozymes CYP1A2, 2E1, and 3A are known to activate AP in the human. In the present study, we determined that olfactory mucosal toxicity of AP was not different between the Cyp1a2(+/+) wild-type and the Cyp1a2(-/-) knockout mouse, whereas the hepatic toxicity of AP was significantly diminished in Cyp1a2(-/-) mice. Western blots of olfactory mucosa revealed that CYP2E1 and CYP3A levels are similar between untreated Cyp1a2(+/+) and Cyp1a2(-/-) mice. Diallyl sulfide (DAS), a known inhibitor of CYP2E1 and of CYP2A10/2A11 (the rabbit orthologue of mouse CYP2A5), completely eliminated olfactory toxicity of AP in both the Cyp1a2(-/-) and wild-type mouse olfactory mucosa. We found that heterologously expressed mouse CYP2A5 and CYP2G1 enzymes (known to be present in olfactory mucosa) form 3-hydroxyacetaminophen (3-OH-AP) and 3-(glutathion-S-yl)acetaminophen (GS-AP); CYP2A5 is considerably more active than 2G1. Addition of GSH caused increases in GS-AP proportional to decreases in 3-OH-AP, suggesting that these two metabolites arise from a common precursor or are formed by way of competing pathways. We also found that both CYP2A5 and CYP2G1 are inhibitable by DAS in vitro. These studies provide strong evidence that, in addition to CYP2E1, CYP2A5 and 2G1 are important in AP bioactivation in the mouse olfactory mucosa and that CYP1A2 appears to be of minor importance for AP olfactory toxicity.

Evaluation of olfactory and auditory system effects of the antihyperthyroid drug carbimazole in the Long-Evans rat.

Carbimazole (2-carbethoxythio-1-methylimidazole) is a thiocarbamide drug used in the treatment of hyperthyroidism in humans. Side effects associated with carbimazole treatment are reported to include impaired taste, impaired olfaction, and hearing loss. The structurally similar antihyperthyroid drug methimazole (1-methyl-2-mercaptoimidazole), also reportedly associated with impaired taste and olfaction in humans, has recently been demonstrated by this laboratory to be an olfactory toxicant by both the oral and intraperitoneal routes of exposure in rodents. A systematic evaluation of sensory system effects of these compounds, either in rodents or humans, is not available in the literature. Male Long-Evans rats were used to evaluate the auditory and olfactory toxicity of carbimazole by two routes of exposure. Histopathological evaluation of nasal cavities from rats administered carbimazole via i.p. and oral routes revealed olfactory mucosal damage and early evidence of repair; a no-observed effect level (NOEL) of 100 mg/kg was observed for orally administered carbimazole. Further, these studies demonstrate evidence for the generation of the olfactory toxic metabolites of carbimazole by the olfactory mucosa itself, as incubation of carbimazole with an olfactory S9 preparation resulted in NADPH-dependent degradation of carbimazole. Evaluation of the auditory startle response in carbimazole-treated rats revealed no deficits, demonstrating that carbimazole does not cause a global loss of hearing in rats.

Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes.

The metabolic activation of two known olfactory mucosal (OM) toxicants, acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined with mouse liver and OM microsomes and purified, heterologously expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both isoforms were active in metabolizing DCBN and AP to metabolites that formed protein adducts. The formation of DCBN- or AP-protein adducts and other AP metabolites, including 3-hydroxy-AP and, in the presence of glutathione, AP-glutathione conjugate, was also detected in OM microsomal reactions and to a much greater extent than in liver microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major roles in mouse OM microsomal metabolic activation of DCBN and AP. Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450 isoforms in OM microsomes. OM microsomal AP and DCBN metabolic activation was inhibited by 5- and 8-methoxsalen, which inhibit both CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM bioactivation of AP and DCBN were ruled out by comparing activities of acetone-treated mice or Cyp1a2(-/-) mice with those of control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known OM-selective toxicity of AP and DCBN. Further analysis of the kinetics of AP and DCBN metabolism by the purified P450s suggested that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas their relative roles in DCBN metabolism may be dose dependent, with CYP2G1 playing more important roles at low substrate concentrations.

Age-related susceptibility to 3,3'-iminodipropionitrile-induced olfactory mucosal damage.

3,3'-Iminodipropionitrile (IDPN) causes degeneration of the olfactory mucosa (OM) in rodents following systemic exposure. Approximately 30% of the OM degenerates in 21-day- and 10-week-old rats following a single 200 or 400 mg/kg intraperitoneal (i.p.) dose of IDPN, whereas 100% olfactory mucosal degeneration occurred in 21-month-old rats. Age-related changes in the flavin-containing monooxygenases (FMOs) or heat shock protein 70 (HSP70) were hypothesized to be responsible for altered olfactory mucosal susceptibility to IDPN. FMO activity in OM was higher than in liver in rats up to 40 weeks of age. Western blots of OM and liver revealed no change in FMO1 protein; however, FMO2, 3, and 5 decreased in olfactory microsomes with age. FMO3 and FMO5 increased in liver microsomes with age. Heat shock protein 70 did not differ between 10-week- and 10-month-old rats in either tissue. The mechanism underlying the increased susceptibility of older rats is likely a complex interaction between the activities of one or more of the enzymes involved in IDPN metabolism in OM and liver.

Effect of methimazole, an FMO substrate and competitive inhibitor, on the neurotoxicity of 3,3'-iminodipropionitrile in male rats.

This study was designed to examine the role of flavin-containing monooxygenase (FMO) on the auditory and vestibular neurotoxicity of 3,3'-iminodipropionitrile (IDPN) using the FMO substrate and competitive inhibitor methimazole (MMI). Specifically, the purpose was to block the FMO-mediated conversion of IDPN to the putative neurotoxic metabolite N-hydroxy3,3'-iminodipropionitrile (HOIDPN). In three separate experiments, adult male Long-Evans hooded rats were administered (ip) saline (vehicle), MMI, IDPN, or HOIDPN individually, or a combination of IDPN and MMI or HOIDPN and MMI. Animals were observed daily for signs of the ECC syndrome (excitation with choreiform and circling movements) for 10 days. One to 2 weeks after exposure, a battery of behavioral tests was used to examine vestibular and auditory function. MMI completely blocked the neurotoxicity associated with a 600 mg/kg dose of IDPN and partially blocked the effects of a 1000 mg/kg dose of IDPN. In contrast, MMI failed to block, and instead increased, the neurotoxicity associated with HOIDPN. These data suggest that FMO-mediated metabolism of IDPN is necessary for the generation of a metabolite responsible for the vestibular and auditory neurotoxicities.

Regulation of gene expression for tyrosine hydroxylase in oxygen sensitive cells by hypoxia.

Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension.

Characterization of the effects of N-hydroxy-IDPN on the auditory, vestibular, and olfactory systems in rats.

The mechanism of neurotoxicity of 3,3'-iminodipropionitrile (IDPN) has been widely debated, with either the parent compound or putative metabolites implicated in various studies. The N-hydroxylated form of IDPN (HO-IDPN) has been reported to cause the excitation with choreiform and circling (ECC) syndrome in rats at doses approximately one-eighth of that required to cause comparable signs in rats treated with IDPN. Because of the similarity of symptoms induced by HO-IDPN and IDPN, we investigated the effect of HO-IDPN on other aspects of the nervous system affected by IDPN, specifically the auditory, vestibular, and olfactory systems. In addition, ECC symptoms were quantified to replicate the previous findings. HO-IDPN was administered ip in saline for 3 consecutive days to two different cohorts of young adult male Sprague-Dawley rats. The first cohort (60, 80, 100, and 120 mg/kg; n = 2/group, except for the 120 mg/kg group, where n = 1) was used in a dose range-finding study. After making the neurobehavioral assessments, animals were sacrificed for olfactory mucosal histopathology. Based on the outcome of the first study, the second cohort (n = 10/group) received saline or HO-IDPN at 100 mg/kg/day for 3 consecutive days. Two animals from each of these groups were sacrificed for olfactory mucosal histopathology; the remaining animals were tested for neurobehavioral effects 3 weeks after the last dose. Animals in the second cohort lost approximately 8% of their pretreatment body weight. All rats receiving the 100 mg/kg/day dose of HO-IDPN (and the rat receiving 120 mg/kg/day) developed the ECC syndrome and signs of vestibular dysfunction within 4 days after the last dose. HO-IDPN caused a large decrease in the acoustic startle response and markedly elevated auditory thresholds at all frequencies tested. The threshold for the ECC syndrome and olfactory mucosal damage was 100 mg/kg. These studies extend previous findings on the neurotoxicity of HO-IDPN and point to the need for determining whether HO-IDPN is an in vivo metabolite of IDPN.

Characterization of olfactory deficits in the rat following administration of 2,6-dichlorobenzonitrile (dichlobenil), 3,3'-iminodipropionitrile, or methimazole.

The histopathology of the olfactory mucosal lesion associated with ip administration of 2,6-dichlorobenzonitrile (dichlobenil) and 3,3'-iminodipropionitrile (IDPN) has been well documented. Whether there is an olfactory deficit associated with the partial loss of the olfactory mucosa (localized around the dorsal medial meatus of the nasal cavity) has yet to be determined. Dichlobenil (100 mg/kg) or IDPN (200 mg/kg) was administered ip to adult male Long-Evans rats previously trained in an olfactory task to find a food pellet buried in approximately 7.5 cm of bedding in a 0.61 x 1.2 x 0.61-m Plexiglass chamber. As a positive control, another group received 300 mg/kg ip of 1-methyl-2-mercaptoimidazole (methimazole), a dosing regimen which destroys nearly all of the olfactory mucosa. All three compounds caused a transient increase in the mean latency to find the pellet, with the magnitude of the effect positively correlated with the extent of the olfactory lesion. In order to determine whether these deficits resulted from olfactory dysfunction or impaired cognitive function (a deficit previously attributed to IDPN exposure), another group of rats was dosed as above and tested in another spatial memory task, the Morris water maze (MWM), which is less dependent upon olfactory function. No performance deficit was detected in the MWM. These data suggest that the transient olfactory deficit in the dichlobenil-, IDPN-, and methimazole-treated rats is attributable to defective olfactory function.

Regulation of ionic conductances and gene expression by hypoxia in an oxygen sensitive cell line.

We have shown that the PC12 cell line is an excellent model system for investigations of the molecular and cellular processes involved in O2-chemosensitivity. We have identified an O2-sensitive K channel in this cell line that mediates membrane depolarization, an increase in intracellular free Ca2+, and dopamine release during hypoxia. We also presented evidence which shows that expression of the gene for tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, is stimulated by reduced O2 tension in PC12 and type I carotid body cells. In addition, we have successfully identified the DNA sequences and trans-acting protein factors that regulate transcription of the TH gene during hypoxia. The mechanisms by which a reduction in O2 tension is transduced into alter cell function including increased gene expression remain unknown. Unpublished results from our laboratory show that the increased TH gene expression during hypoxia does not require activation of the cAMP-PKA signal transduction pathway. We propose that the increase in intracellular free Ca2+ that occurs as a result of membrane depolarization might play an important role. Preliminary findings from our laboratory show that blockade of the voltage operated Ca2+ channel or chelation of intracellular Ca2+ prevent full activation of the TH gene during hypoxia.

Distribution of microsomal epoxide hydrolase and glutathione S-transferase in the rat olfactory mucosa: relevance to distribution of lesions caused by systemically-administered olfactory toxicants.

This study represents part an of ongoing effort to understand the mechanism underlying the distribution of the olfactory mucosal lesion resulting from the systemic administration of compounds such as 2,6-dichlorobenzonitrile (dichlobenil) and beta,beta'-iminodipropionitrile (IDPN). Immunohistochemistry was performed to localize the microsomal form of epoxide hydrolase (mEH) and glutathione S-transferase (GST) isozymes alpha, mu and pi in the rodent olfactory mucosa. GST-pi was found in abundance in the Bowman's glands of the mucosa lining the dorsal medial meatus (DMM) of the nasal cavity and in the nuclei of basal and sustentacular cells of the dorsal and lateral nasal cavity. Liver and olfactory mucosal levels of mEH are equivalent by Western blot analysis. mEH appeared to be localized in the apical cytoplasm of sustentacular cells in all regions of the olfactory mucosa except for the epithelium lining the DMM. These observations, coupled with the known profile of metabolites for dichlobenil, suggest that systemically-administered compounds causing site-specific lesions in the epithelium lining the DMM of the nasal cavity may do so by the in situ production of reactive epoxide metabolites which are then poorly capable of being detoxified. Thus, the distribution of metabolic enzymes, rather than the absolute level of an enzyme in a tissue, may dictate lesion distribution in the case of toxicants which are bioactivated in target tissues.