Olfactory Toxicity Research Articles

Differential response of olfactory sensory neuron populations to copper ion exposure in zebrafish

The peripheral olfactory system of fish is in direct contact with the external aqueous environment, so dissolved contaminants can easily impair sensory functions and cause neurobehavioral injuries. The olfactory epithelium of fish is arranged in lamellae forming a rosette in the olfactory cavity and contains three main types of olfactory sensory neurons (OSNs): ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs), common to all vertebrates, and a third minor group of olfactory neurons, crypt cells, absent in tetrapods. Since copper is a ubiquitously diffusing olfactory toxicant and a spreading contaminant in urban runoff, we investigated the effect of low copper concentration on the three different OSNs in the olfactory epithelium of zebrafish, a model system widely used in biological research. Image analysis was applied for morphometry and quantification of immunohistochemically detected OSNs. Copper exposure resulted in an evident decrease in olfactory epithelium thickness. Moreover, after exposure, the lamellae of the dorsal and ventral halves of the olfactory rosettes showed a different increase in their sensory areas, suggesting a lateral migration of new cells into non-sensory regions. The results of the present study provide clear evidence of a differential response of the three neural cell populations of zebrafish olfactory mucosa after 96h of exposure to copper ions at the sublethal concentration of 30μgL−1. Densitometric values of cONS, immunostained with anti-G αolf, decreased of about 60% compared to the control. When the fish were transferred to water without copper addition and examined after 3, 10 and 30days, we observed a partial restoration of anti-G αolf staining intensity to normal condition. The recovery of cOSNs appeared sustained by neuronal proliferation, quantified with anti-PCNA immunostaining, in particular in the early days after exposure. The densitometric analysis applied to mOSNs, immunostained with anti-TRPC2, revealed a statistically significant decrease of about 30% compared to the control. For cOSNs and mOSNs, the decrement in staining intensity may be indicative of cell death, but reduction in antigen expression may not be excluded. In the post-exposure period of 1 month we did not find recovery of mOSNs. We hypothesize that cOSNs are more sensitive than mOSNs to copper treatment, but also more prompted to tissue repair. Anti-TrkA-immunopositive crypt cells appeared not to be affected by copper exposure since statistical analysis excluded any significant difference between the control and treated fish. Comparative studies on OSNs would greatly enhance our understanding of the mechanisms of olfaction.

Exposure to Zinc Sulfate Results in Differential Effects on Olfactory Sensory Neuron Subtypes in Adult Zebrafish.

Zinc sulfate is a known olfactory toxicant, although its specific effects on the olfactory epithelium of zebrafish are unknown. Olfactory organs of adult zebrafish were exposed to zinc sulfate and, after 2, 3, 5, 7, 10 or 14 days, fish were processed for histological, immunohistochemical, ultrastructural, and behavioral analyses. Severe morphological disruption of the olfactory organ was observed two days following zinc sulfate exposure, including fusion of lamellae, epithelial inflammation, and significant loss of anti-calretinin labeling. Scanning electron microscopy revealed the apical surface of the sensory region was absent of ciliated structures, but microvilli were still present. Behavioral analysis showed significant loss of the ability to perceive bile salts and some fish also had no response to amino acids. Over the next several days, olfactory organ morphology, epithelial structure, and anti-calretinin labeling returned to control-like conditions, although the ability to perceive bile salts remained lost until day 14. Thus, exposure to zinc sulfate results in rapid degeneration of the olfactory organ, followed by restoration of morphology and function within two weeks. Zinc sulfate appears to have a greater effect on ciliated olfactory sensory neurons than on microvillous olfactory sensory neurons, suggesting differential effects on sensory neuron subtypes.

Olfactory toxicity in fish-Why we should care about it.

Olfactory toxicity in fish-Why we should care about it.

Effects of salinity on olfactory toxicity and behavioral responses of juvenile salmonids from copper

Dissolved copper is one of the more pervasive and toxic constituents of stormwater runoff and is commonly found in stream, estuary, and coastal marine habitats of juvenile salmon. While stormwater runoff does not usually carry copper concentrations high enough to result in acute lethality, they are of concern because sublethal concentrations of copper exposure have been shown to both impair olfactory function and alter behavior in various species in freshwater. To compare these results to other environments that salmon are likely to encounter, experiments were conducted to evaluate the effects of salinity on the impairment of olfactory function and avoidance of copper. Copper concentrations well within the range of those found in urban watersheds, have been shown to diminish or eliminate the olfactory response to the amino acid, l-serine in freshwater using electro-olfactogram (EOG) techniques. The olfactory responses of both freshwater-phase and seawater-phase coho and seawater-phase Chinook salmon, were tested in freshwater or seawater, depending on phase, and freshwater-phase coho at an intermediate salinity of 10‰. Both 10‰ salinity and full strength seawater protected against the effects of 50μg copper/L. In addition to impairing olfactory response, copper has also been shown to alter salmon behavior by causing an avoidance response. To determine whether copper will cause avoidance behavior at different salinities, experiments were conducted using a multi-chambered experimental tank. The circular tank was divided into six segments by water currents so that copper could be contained within one segment yet fish could move freely between them. The presence of individual fish in each of the segments was counted before and after introduction of dissolved copper (<20μg/L) to one of the segments in both freshwater and seawater. To address whether use of preferred habitat is altered by the presence of copper, experiments were also conducted with a submerged structural element. The presence of sub-lethal levels of dissolved copper altered the behavior of juvenile Chinook salmon by inducing an avoidance response in both freshwater and seawater. While increased salinity is protective against loss of olfactory function from dissolved copper, avoidance could potentially affect behaviors beneficial to growth, survival and reproductive success.

Hyposmia: an underestimated and frequent adverse effect of chemotherapy.

Optimal function of both the olfactory sensory neurons and the olfactory mucosa is a prerequisite for normal olfactory perception. Both the olfactory neurons and mucosa might be subjects to the neurotoxic and mucotoxic effects of chemotherapy. Despite the recognized importance of olfaction in nutrition and quality of life, the potential olfactory toxicity of chemotherapy regimens has not been adequately assessed. The aim of this study is to investigate whether mucotoxic and/or neurotoxic drugs compromise olfactory performance. Forty-four consecutive patients completed the "Sniffin' Sticks" test, an objective quantitative/qualitative method to assess olfactory function, at diagnosis and immediately before the infusion of the last session of three to four chemotherapy cycles, according to the therapeutic protocol. The patients underwent therapy containing oxaliplatin and antimetabolites (5-FU or capecitabine; O+A group), taxanes and platinum analogues (cisplatin and carboplatin; T+P group), or taxanes and anthracyclines (doxorubicin or liposomal doxorubicin; T+A group). A significant decrease was noted for olfactory threshold (OT), olfactory discrimination (OD), olfactory identification (OI), and the composite threshold-discrimination-identification (TDI) score. A significant deterioration of all olfactory indices was found for each chemotherapy group. Pairwise comparisons revealed significant differences between the O+A and the T+P group regarding OT and TDI. TDI scores were significantly lower after chemotherapy in all age groups. Patients older than 50 years were found to be more susceptible to olfactory toxicity than younger patients. Patients who undergo chemotherapy experience significant compromise in their olfactory function. A grading system for olfactory toxicity is proposed.

Olfactory mucosal necrosis in rats following acute intraperitoneal administration of 1,2-diethylbenzene, 1,2-diacetylbenzene and 2,5-hexanedione

1,2-Diethylbenzene (1,2-DEB) is used in the manufacture of some plastics. Exposure to 1,2-DEB has been shown to induce peripheral neuropathy in rats. This neurotoxicity is thought to be caused by a metabolite, 1,2-diacetylbenzene (1,2-DAB), a γ-diketone-like compound. 1,2-DEB was previously shown to be extensively and rapidly taken up by the nasal mucosa in male rats. In the present study, the nasal mucosa in rats exposed to 1,2-DEB and 1,2-DAB were examined histologically. Results were compared to sections from rats exposed to two other DEB isomers – 1,3-diethylbenzene (1,3-DEB) and 1,4-diethylbenzene (1,4-DEB) – and to two other neurotoxic compounds – n-hexane and its γ-diketone metabolite, 2,5-hexanedione (2,5-HD). A single intraperitoneal dose of 1,2-DEB (200mg/kg) induced time-dependent necrosis in the olfactory epithelium and Bowman's glands, with lesions appearing from the earliest observation time (4h) in the dorsomedial olfactory mucosa. Lesions spread through the lateral and ventral parts of the ethmoturbinates over the following days. The dorsal and medial zones of the nasal cavity started to regenerate from 72h after treatment, with the new epithelium showing metaplasia. One month after treatment, most of the olfactory epithelium had returned to normal. 1,2-DAB (40mg/kg) caused the same lesions as those observed after treatment with 1,2-DEB. Treatment with 2,5-HD (1g/kg) also caused lesions of the olfactory epithelium, mainly at level IV. However, these were comparatively less severe than those observed after exposure to 1,2-DEB. In contrast, intraperitoneal injection of 1,3-DEB (800mg/kg), 1,4-DEB (800mg/kg) and n-hexane (2g/kg) did not affect the nasal mucosa. Pretreatment of rats with 5-phenyl-1-pentyne, an inhibitor of CYP2F2 and CYP2E1 completely inhibited the olfactory toxicity caused by 1,2-DEB. These results suggest that metabolic activation of 1,2-DEB may be responsible for the toxicity observed.

Mechanisms of permanent loss of olfactory receptor neurons induced by the herbicide 2,6-dichlorobenzonitrile: Effects on stem cells and noninvolvement of acute induction of the inflammatory cytokine IL-6

We explored the mechanisms underlying the differential effects of two olfactory toxicants, the herbicide 2,6-dichlorobenzonitrile (DCBN) and the anti-thyroid drug methimazole (MMZ), on olfactory receptor neuron (ORN) regeneration in mouse olfactory epithelium (OE). DCBN, but not MMZ, induced inflammation-like pathological changes in OE, and DCBN increased interleukin IL-6 levels in nasal-wash fluid to much greater magnitude and duration than did MMZ. At 24h after DCBN injection, the population of horizontal basal cells (HBCs; reserve, normally quiescent OE stem cells) lining the DMM became severely depleted as some of them detached from the basal lamina, and sloughed into the nasal cavity along with the globose basal cells (GBCs; heterogeneous population of stem and progenitor cells), neurons, and sustentacular cells of the neuroepithelium. In contrast, the layer of HBCs remained intact in MMZ-treated mice, as only the mature elements of the neuroepithelium were shed. Despite the respiratory metaplasia accompanying the greater severity of the DCBN lesion, residual HBCs that survived intoxication were activated by the injury and contributed to the metaplastic respiratory epithelium, as shown by tracing their descendants in a K5CreErT2::fl(stop)TdTomato strain of mice in which recombination causes HBCs to express TdTomato in advance of the lesion. But, contrary to published observations with MMZ, the HBCs failed to form ORNs. A role for IL-6 in suppressing ORN regeneration in DCBN-treated mice was rejected by the failure of the anti-inflammatory drug dexamethasone to prevent the subsequent respiratory metaplasia in the DMM, suggesting that other factors lead to HBC neuro-incompetence.

Role of Nrf2 antioxidant defense in mitigating cadmium-induced oxidative stress in the olfactory system of zebrafish

Exposure to trace metals can disrupt olfactory function in fish leading to a loss of behaviors critical to survival. Cadmium (Cd) is an olfactory toxicant that elicits cellular oxidative stress as a mechanism of toxicity while also inducing protective cellular antioxidant genes via activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, the molecular mechanisms of Cd-induced olfactory injury have not been characterized. In the present study, we investigated the role of the Nrf2-mediated antioxidant defense pathway in protecting against Cd-induced olfactory injury in zebrafish. A dose-dependent induction of Nrf2-regulated antioxidant genes associated with cellular responses to oxidative stress was observed in the olfactory system of adult zebrafish following 24h Cd exposure. Zebrafish larvae exposed to Cd for 3h showed increased glutathione S-transferase pi (gst pi), glutamate–cysteine ligase catalytic subunit (gclc), heme oxygenase 1 (hmox1) and peroxiredoxin 1 (prdx1) mRNA levels indicative of Nrf2 activation, and which were blocked by morpholino-mediated Nrf2 knockdown. The inhibition of antioxidant gene induction in Cd-exposed Nrf2 morphants was associated with disruption of olfactory driven behaviors, increased cell death and loss of olfactory sensory neurons (OSNs). Nrf2 morphants also exhibited a downregulation of OSN-specific genes after Cd exposure. Pre-incubation of embryos with sulforaphane (SFN) partially protected against Cd-induced olfactory tissue damage. Collectively, our results indicate that oxidative stress is an important mechanism of Cd-mediated injury in the zebrafish olfactory system. Moreover, the Nrf2 pathway plays a protective role against cellular oxidative damage and is important in maintaining zebrafish olfactory function.

Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase (gpx4) isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium

Exposure to environmental contaminants, including various pesticides and trace metals, can disrupt critical olfactory-driven behaviors of fish such as homing to natal streams, mate selection, and an ability to detect predators and prey. These neurobehavioral injuries have been linked to reduced survival and population declines. Despite the importance of maintaining proper olfactory signaling processes in the presence of chemical exposures, little is known regarding chemical detoxification in the salmon olfactory system, and in particular, the antioxidant defenses that maintain olfactory function. An understudied, yet critical component of cellular antioxidant defense is phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4), an isoform within the family of selenium-dependent glutathione peroxidase (GPx) enzymes that can directly reduce lipid peroxides and other membrane-bound complex hydroperoxides. In this study, we cloned two gpx4 isoforms (gpx4a and gpx4b) from Coho salmon olfactory tissues and compared their modulation in olfactory and liver tissues by cadmium, an environmental pollutant and olfactory toxicant that cause oxidative damage as a mechanism of toxicity. Amino acid sequence comparisons of the two gpx4 isoforms shared 71% identity, and also relatively high sequence identities when compared with other fish GPx4 isoforms. Sequence comparisons with human GPx4 indicated conservation of three important active sites at selenocysteine (U46), glutamine (Q81), and tryptophan (W136), suggesting similar catalytic activity between fish and mammalian GPx4 isoforms. Tissue profiling confirmed the expression of gpx4a and gpx4b in all ten Coho tissues examined. The expression of gpx4 mRNAs in the Coho olfactory system was accompanied by comparably high initial rates of GPx4 enzymatic activity in mitochondrial and cytosolic fractions. Exposure to low (3.7ppb) and high (347ppb) environmental Cd concentrations for 24–48h significantly decreased gpx4a expression in Coho olfactory rosettes, whereas olfactory gpx4b mRNA expression was not modulated by exposures at these concentrations. In summary, Coho salmon express two paralogs of gpx4, a key enzyme in the maintenance of signal transduction processes that protect against cellular oxidative damage. The Cd-associated downregulation of salmon olfactory gpx4a expression in particular, may be associated with the loss of olfactory signal transduction that accompanies metal-associated loss of olfaction in salmonids.

Silver nanoparticles disrupt olfaction in Crucian carp ( Carassius carassius) and Eurasian perch ( Perca fluviatilis)

The present study investigates the effect of silver nanoparticles on olfaction in Crucian carp ( Carassius carassius) and Eurasian perch ( Perca fluviatilis). The electro-olfactogram (EOG) signal was recorded by stimulating the olfactory epithelium with pulses of the odorant l-alanine during the pre-exposure, silver exposure and recovery periods, respectively. The nanosilver suspension concentrations applied were 0.00, 0.45 and 45 μg L −1, respectively. Secondly, to compare the toxicity of silver nanoparticles with silver ions, perch were exposed to ionic silver. During exposure to nanosilver suspension, the olfactory epithelium rapidly hyperpolarized, which was not found after exposure to silver ion solution. Exposure to 0.45 μg L −1 nanosilver suspension led to enhanced EOG responses, whereas exposure to 45 μg L −1 silver nanoparticle suspension and silver ion solution resulted in suppressed EOG signals. The EOG signals partly recovered in silver-free water. The silver nanoparticle olfactory toxicity is believed to be a combination of silver particles and released silver ions.

Mechanisms of olfactory toxicity of the herbicide 2,6-dichlorobenzonitrile: Essential roles of CYP2A5 and target-tissue metabolic activation

The herbicide 2,6-dichlorobenzonitril (DCBN) is a potent and tissue-specific toxicant to the olfactory mucosa (OM). The toxicity of DCBN is mediated by cytochrome P450 (P450)-catalyzed bioactivation; however, it is not known whether target-tissue metabolic activation is essential for toxicity. CYP2A5, expressed abundantly in both liver and OM, was previously found to be one of the P450 enzymes active in DCBN bioactivation in vitro. The aims of this study were to determine the role of CYP2A5 in DCBN toxicity in vivo, by comparing the extents of DCBN toxicity between Cyp2a5-null and wild-type (WT) mice, and to determine whether hepatic microsomal P450 enzymes (including CYP2A5) are essential for the DCBN toxicity, by comparing the extents of DCBN toxicity between liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, and WT mice. We show that the loss of CYP2A5 expression did not alter systemic clearance of DCBN (at 25 mg/kg); but it did inhibit DCBN-induced non-protein thiol depletion and cytotoxicity in the OM. Thus, CYP2A5 plays an essential role in mediating DCBN toxicity in the OM. In contrast to the results seen in the Cyp2a5-null mice, the rates of systemic DCBN clearance were substantially reduced, while the extents of DCBN-induced nasal toxicity were increased, rather than decreased, in the LCN mice, compared to WT mice. Therefore, hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. Our findings form the basis for a mechanism-based approach to assessing the potential risks of DCBN nasal toxicity in humans.

Copper-induced olfactory toxicity in salmon and steelhead: Extrapolation across species and rearing environments

Recent research has shown that hatchery coho salmon ( Oncorhynchus kisutch) are vulnerable to the olfactory neurotoxicity caused by copper from urban runoff, pesticide use, and mining activities. To explore the broader application of this data to salmonids living in the wild, we exposed naturally-reared steelhead ( O. mykiss) to copper (5 and 20 μg/L; 3 h) and measured losses in olfactory function via electro-olfactogram (EOG) recordings. Copper exposure disrupted the olfactory responsiveness of steelhead to an amino acid ( l-serine) in a dose-dependent manner that was equivalent to previously published data for hatchery coho. Our findings support extrapolation of copper toxicity data across species and from fish raised in hatcheries to fish in the wild.

Bioactivation of coumarin in rat olfactory mucosal microsomes: Detection of protein covalent binding and identification of reactive intermediates through analysis of glutathione adducts

The presence of high levels, as well as tissue-specific forms, of cytochrome P450 enzymes in mammalian olfactory mucosa (OM) has important implications in the bioactivation and toxicity of xenobiotics entering the tissue. Previous studies have shown that coumarin, a known olfactory toxicant in rats, is bioactivated by OM microsomal P450s to a number of products, presumably via coumarin-3,4-epoxide and other epoxide intermediates. The aim of the current study was to obtain direct evidence for the formation of such reactive intermediates in rat OM through the detection of protein covalent binding and glutathione (GSH) adduct formation. Protein covalent binding experiments with [ 14C]coumarin (10 μM) displayed a 7–9-fold higher NADPH-dependent radioactivity binding in rat OM microsomes (2.5 nmol/mg/30 min) compared to those in rat and human liver microsomes; the binding value in rat OM microsomes was substantially but not completely reduced by the addition of GSH (5 mM). LC/MS analyses detected a number of GSH adducts in GSH-supplemented coumarin metabolism reaction in rat OM microsomes; 3-glutathionyl coumarin was found to be the major one, indicating 3,4-epoxidation as the main bioactivation pathway. Additional GSH adducts were identified, presumably forming via the same pathway or epoxidation on the benzene moiety. Our findings provide direct evidence for the formation of multiple coumarin reactive intermediates in rat OM, leading to protein covalent binding and GSH conjugation.

Chemosensory Deprivation in Juvenile Coho Salmon Exposed to Dissolved Copper under Varying Water Chemistry Conditions

Dissolved copper is an important nonpoint source pollutant in aquatic ecosystems worldwide. Copper is neurotoxic to fish and is specifically known to interfere with the normal function of the peripheral olfactory nervous system. However,the influence of water chemistry on the bioavailability and toxicity of copper to olfactory sensory neurons is not well understood. Here we used electrophysiological recordings from the olfactory epithelium of juvenile coho salmon (Oncorhynchus kisutch) to investigate the impacts of copper in freshwaters with different chemical properties. In low ionic strength artificial fresh water, a short-term (30 min) exposure to 20 microg/L dissolved copper reduced the olfactory response to a natural odorant (10(-5) M L-serine) by 82%. Increasing water hardness (0.2-1.6 mM Ca) or alkalinity (0.2-3.2 mM HCO3-) only slightly diminished the inhibitory effects of copper. Moreover, the loss of olfactory function was not affected by a change in pH from 8.6 to 7.6. By contrast, olfactory capacity was partially restored by increasing dissolved organic carbon (DOC; 0.1-6.0 mg/L). Given the range of natural water quality conditions in the western United States, water hardness and alkalinity are unlikelyto protect threatened or endangered salmon from the sensory neurotoxicity of copper. However, the olfactory toxicity of copper may be partially reduced in surface waters that have a high DOC content.

Evaluation of Long-Term Occupational Exposure to Styrene Vapor on Olfactory Function

The primary sensory neurons of the olfactory system are chronically exposed to the ambient environment and may therefore be susceptible to damage from occupational exposure to many volatile chemicals. To investigate whether occupational exposure to styrene was associated with olfactory impairment, we examined olfactory function in 2 groups: workers in a German reinforced-plastics boat-manufacturing facility having a minimum of 2 years of styrene exposure (15-25 ppm as calculated from urinary metabolite concentrations, with historical exposures up to 85 ppm) and a group of age-matched workers from the same facility with lower styrene exposures. The results were also compared with normative data previously collected from healthy, unexposed individuals. Multiple measures of olfactory function were evaluated using a standardized battery of clinical assessments from the Monell-Jefferson Chemosensory Clinical Research Center that included tests of threshold sensitivity for phenylethyl alcohol (PEA) and odor identification ability. Thresholds for styrene were also obtained as a measure of occupational olfactory adaptation. Styrene exposure history was calculated through the use of past biological monitoring results for urinary metabolites of styrene (mandelic acid [MA], phenylglyoxylic acid [PGA]); current exposure was determined for each individual using passive air sampling for styrene and biological monitoring for styrene urinary metabolites. Current mean effective styrene exposure during the day of olfactory testing for the group of workers who worked directly with styrene resins was 18 ppm styrene (standard deviation [SD] = 14), 371 g/g creatinine MA + PGA (SD = 289) and that of the group of workers with lower exposures was 4.8 ppm (SD = 5.2), 93 g/g creatinine MA+PGA (SD = 100). Historic annual average exposures for all workers were greater by a factor of up to 6x. No differences unequivocally attributable to exposure status were observed between the Exposed and Comparison groups or between performance of either group and normative population values on thresholds for PEA or odor identification. Although odor identification performance was lower among workers with higher ongoing exposures, performance on this test is not a pure measure of olfactory ability and is influenced by familiarity with the stimuli and their sources. Consistent with exposure-induced sensory adaptation, however, elevated styrene thresholds were significantly associated with higher occupational exposures to styrene. In summary, the present study found no evidence among a cross-section of reinforced-plastics workers that current or historical exposure to styrene was associated with a general impairment of olfactory function. When taken together with prior studies of styrene-exposed workers, these results suggest that styrene is not a significant olfactory toxicant in humans at current exposure levels.

Differential gene expression in the olfactory bulb following exposure to the olfactory toxicant 2,6-dichlorophenyl methylsulphone and its 2,5-dichlorinated isomer in mice

2,6-Dichlorophenyl methylsulphone and a number of structurally related chemicals are CYP-activated toxicants in the olfactory mucosa in mice and rats. This toxicity involves both the olfactory neuroepithelium and its subepithelial nerves. In addition, 2,6-dichlorophenyl methylsulphone induces glial acidic fibrillary protein expression (Gfap, a biomarker for gliosis) in the olfactory bulb, as well as long-lasting learning deficits and changes in spontaneous behavior in mice and rats. So far the 2,5-dichlorinated isomer has not been reported to cause toxicity in the olfactory system, although it gives rise to transient changes in spontaneous behavior. In the present study we used 15k cDNA gene arrays and real-time RT-PCR to determine 2,6-dichlorophenyl methylsulphone-induced effects on gene expression in the olfactory bulb in mice. Seven days following a single ip dose of 2,6-dichlorophenyl methylsulphone, 56 genes were found to be differentially expressed in the olfactory bulb. Forty-one of these genes clustered into specific processes regulating, for instance, cell differentiation, cell migration and apoptosis. The genes selected for real-time RT-PCR were chosen to cover the range of B-values in the cDNA array analysis. Altered expression of Gfap, mt-Rnr2, Ncor1 and Olfml3 was confirmed. The expression of these genes was measured also in mice dosed with 2,5-dichlorophenyl methylsulphone, and mt-Rnr2 and Olfml3 were found to be altered also by this isomer. Combined with previous data, the results support the possibility that the persistent neurotoxicity induced by 2,6-dichlorophenyl methylsulphone in mice represents both an indirect and a direct effect on the brain. The 2,5-dichlorinated isomer, negative with regard to CYP-catalyzed toxicity in the olfactory mucosa, may prove useful to resolve this issue.

Naphthalene toxicity in mice and aryl hydrocarbon receptor-mediated CYPs

Naphthalene (NP) has been designated a ‘reasonably anticipated human carcinogen’ because of positive responses in carcinogenicity bioassays in rodents. Whereas CYP2F enzymes are widely regarded as responsible for NP bioactivation, other metabolic enzymes—including CYP1A1 and CYP1A2—produce NP-1,2-oxide in vitro. We investigated the role of these aryl hydrocarbon receptor (AHR)-mediated enzymes in NP toxicity in two ways. First, NP was assessed for the ability to activate transcription via the AHR in an in vitro luciferase reporter assay and was found to have no activity. Second, mice deficient in AHR, CYP1A1 or CYP1A2 were dosed with NP alone, or following pretreatment with the CYP2F inhibitor 5-phenyl-1-pentyne. None of the knockout mice were protected from olfactory toxicity of NP. In contrast, CYP1A1- and CYP1A2-null mice pretreated with 5-phenyl-1-pentyne exhibited no NP olfactory toxicity. These results suggest that AHR-mediated enzymes do not contribute significantly to NP bioactivation in the intact animal.

Olfactory toxicity: long-term effects of occupational exposures

To present and discuss the results of research on olfactory function impairments related to chronic occupational exposure to industrial chemicals. This review is mainly focused on the results of epidemiological studies on olfactory function, evaluated using quantitative testing methods, in workers chronically exposed to airborne industrial chemicals. Papers published in peer-reviewed scientific journals were mainly considered. The prevalence of olfactory impairments related to occupational exposure to chemicals is unknown: frequencies ranging 0.5-5% of all olfactory dysfunctions have been proposed, considering both exposure to chemicals and the use of pharmaceutical drugs, but the real relevance of this problem is possibly overlooked, especially considering that occupational exposure may account for a significant part of "idiopathic" smell disorders, i.e., the 10-25% of all olfactory problems within the general population. An adverse effect has been reported in workers chronically exposed to some metals as cadmium, chromium, manganese, arsenic, mercury, and organic lead, and to other chemicals as acrylates, styrene, and solvent mixtures. The results of relevant studies are discussed. A problem in the evaluation of data is that different methods have been applied in different studies, affecting the comparability of results. To date, knowledge of the effect of chronic occupational exposure to industrial chemicals on olfactory function is largely incomplete, but supports the hypothesis that olfactory neuroepithelium is susceptible to environmental exposures to chemicals. Occupational-related olfactory impairment is usually sub-clinical, and can be only detected using adequate quantitative olfactory function testing procedures. Available data show the need for further good quality research in this field.

CYP2A5-MEDIATED ACTIVATION AND EARLY ULTRASTRUCTURAL CHANGES IN THE OLFACTORY MUCOSA: STUDIES ON 2,6-DICHLOROPHENYL METHYLSULFONE

2,6-Dichlorophenyl methylsulfone (2,6-diClPh-MeSO2) is a potent olfactory toxicant reported to induce endoplasmic reticulum (ER) stress, caspase activation, and extensive cell death in mice. The aim of the present study was to examine cytochrome P450 (P450)-dependent bioactivation, nonprotein sulfhydryl (NP-SH) levels, and early ultrastructural changes in mouse olfactory mucosa following an i.p. injection of 2,6-diClPh-MeSO2 (32 mg/kg). A high covalent binding of 2,6-diClPh-14C-MeSO2 in olfactory mucosa S9 fraction was observed, and the CYP2A5/CYP2G1 substrates coumarin and dichlobenil significantly decreased the binding, whereas the CYP2E1 substrate chlorzoxazone had no effects. An increased bioactivation was detected in liver microsomes of mice pretreated with pyrazole, known to induce CYP2A4, 2A5, 2E1, and 2J, and addition of chlorzoxazone reduced this binding. 2,6-DiClPh-14C-MeSO2 showed a marked covalent binding to microsomes of recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 compared with wild type. One and 4 h after a single injection of 2,6-diClPh-MeSO2, the NP-SH levels in the olfactory mucosa were significantly reduced compared with control, whereas there was no change in the liver. Ultrastructural studies revealed that ER, mitochondria, and secretory granules in nonneuronal cells were early targets 1 h after injection. We propose that lesions induced by 2,6-diClPh-MeSO2 in the mouse olfactory mucosa were initiated by a P450-mediated bioactivation in the Bowman's glands and depletion of NP-SH levels, leading to disruption of ion homeostasis, organelle swelling, and cell death. The high expression of CYP2A5 in the olfactory mucosa is suggested to play a key role for the tissue-specific toxicity induced by 2,6-diClPh-MeSO2.

Toxicant-induced ER-stress and caspase activation in the olfactory mucosa

The potent olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO(2)) induces rapid cell death and long-term metaplastic changes in the olfactory regions of rodents. The damage is related to a tissue-specific and extensive cytochrome P450 (CYP)-mediated metabolic activation of the compound to reactive intermediates. The aim of the present study was to examine the early, cell-specific changes leading to cell death in the olfactory mucosa of mice exposed to 2,6-diClPh-MeSO(2). We have examined the expression of the ER-specific stress protein GRP78, the presence of secretory glycoproteins, and the cellular activation of the initiator caspase 12 and the downstream effector caspase 3. 2,6-DiClPh-MeSO(2) induced rapid and cell-specific expression of GRP78, and activation of caspases 12 and 3 in the Bowman's glands. No similar early onset changes in the neuroepithelium were observed. Based on these results, we propose that extensive lesions are initiated in the Bowman's glands and that the metabolic activation of 2,6-diClPh-MeSO(2) elicits ER-stress response and subsequent apoptotic signaling at this site. Since most of the Bowman's glands had oncotic morphology, the results suggest that the terminal phase of apoptosis was blocked and that these glands finally succumb to other routes of cell death.