Vanadium exposure induces olfactory dysfunction in an animal model of metal neurotoxicity

Abstract

Epidemiological evidence indicates chronic environmental exposure to transition metals may play a role in chronic neurodegenerative conditions such as Parkinson's disease (PD). Chronic inhalation exposure to welding fumes containing metal mixtures may be associated with development of PD. A significant amount of vanadium is present in welding fumes, as vanadium pentoxide (V2O5), and incorporation of vanadium in the production of high strength steel has become more common. Despite the increased vanadium use in recent years, the neurotoxicological effects of this metal are not well characterized. Recently, we demonstrated that V2O5 induces dopaminergic neurotoxicity via protein kinase C delta (PKCδ)-dependent oxidative signaling mechanisms in dopaminergic neuronal cells. Since anosmia (inability to perceive odors) and non-motor deficits are considered to be early symptoms of neurological diseases, in the present study, we examined the effect of V2O5 on the olfactory bulb in animal models. To mimic the inhalation exposure, we intranasally administered C57 black mice a low-dose of 182μg of V2O5 three times a week for one month, and behavioral, neurochemical and biochemical studies were performed. Our results revealed a significant decrease in olfactory bulb weights, tyrosine hydroxylase (TH) levels, levels of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) and increases in astroglia of the glomerular layer of the olfactory bulb in the treatment groups relative to vehicle controls. Neurochemical changes were accompanied by impaired olfaction and locomotion. These findings suggest that nasal exposure to V2O5 adversely affects olfactory bulbs, resulting in neurobehavioral and neurochemical impairments. These results expand our understanding of vanadium neurotoxicity in environmentally-linked neurological conditions.

Keywords: Neurotoxicity; Non-motor symptoms; Olfactory system; Parkinson's disease; Risk assessment; Vanadium.

Fig. 1. Effects of intranasally administered vanadium on locomotor activity

Male C57 black mice were intranasally administered 182 µg of V₂O₅ in 50 µL of de-ionized water three times a week for one month. The vehicle control animals were administered de-ionized water. Locomotor activity was measured using an automated VersaMax locomotor activity monitor before the termination of the study. A, Representative moving tracks of mice. B, Total vertical movement. C, Total horizontal movement. D, Total distance travelled. E, Total movement time. F, Total rest time. The vehicle-treated group served as the control. Data are in percent control and represent mean ± S.E.M. from at least five animals per group. Asterisks (*, p < 0.05; **, p<0.001) indicate significant differences between treatment and the control group.

Fig. 2. Effects of intranasally administered vanadium on pheromonal olfaction (sniffing ability)

Male C57 black mice (n ≥ 5 per group) were intranasally administered 182 µg of V₂O₅ in 50 µL of deionized water three times a week for one month. The vehicle control animals were administered deionized water. At the end of the study, animals were exposed to bedding from pregnant female cages, and the amount of time males spent sniffing the bedding during a five-minute time period was recorded. Asterisks (***, p < 0.001) indicate a significant difference between treatment and control group means ± S.E.M.

Fig. 3. Effects of intranasally administered vanadium on the size of olfactory lobes

Male C57 black mice (n ≥ 5 per group) were intranasally administered 182 µg of V₂O₅ in 50 µL of deionized water three times a week for one month. The vehicle control animals were administered deionized water. At the end of treatment, olfactory lobes were dissected out and weighed. Asterisks (*, p < 0.05) indicate a significant difference between treatment and control group means ± S.E.M.

Fig. 4. Effect of intranasally administered vanadium on TH expression levels in olfactory lobes

Male C57 black mice (n ≥ 3 per group) were intranasally administered 182 µg of V₂O₅ in 50 µL of deionized water three times a week for one month. The vehicle control animals were administered deionized water. At the end of the treatment animals were sacrificed and TH expression was detected in the olfactory lobe by Western blot using mouse monoclonal antibody against TH. (A) A representative Western blot analysis of TH expression in control and vanadium treated mice. β-actin immunoblot was used to confirm equal protein loading in each lane. (B) The bands were quantified for densitometric analysis and data are expressed as a percentage of vehicle-treated bands. Asterisks (***, p < 0.001) indicate a significant difference between treatment and control group means ± S.E.M.

Fig. 5. Loss of glomerular TH neurons in olfactory lobes following intranasally administered vanadium

Male C57 black mice were intranasally administered 182 µg of V₂O₅ in 50 µL of de-ionized water three times a week as described in methods. The vehicle control animals were administered de-ionized water. At the end of treatment, animals were intracardially perfused and TH neurons were detected in the glomerular layer of the olfactory lobe by immunohistochemistry. (A) Representative 2× pictures of TH neurons in the glomerular layer of the olfactory lobe of control and vanadium-treated mice. (B) Representative 30× pictures of TH neurons in the glomerular layer of the olfactory lobe of control and vanadium-treated mice. (C) Representative 60× pictures of TH neurons in the glomerular layer of the olfactory lobe of control and vanadium-treated mice.

Fig. 6. Effects of intranasally administered vanadium on olfactory bulb dopamine and DOPAC levels

Male C57 black mice (n ≥ 5 per group) were intranasally administered 182 µg of V₂O₅ in 50 µL of deionized water three times a week for one month. The vehicle control animals were administered deionized water. Animals were sacrificed following the last treatment, and the neurochemical analysis (A, dopamine; B, DOPAC) was performed in olfactory lobe tissues using HPLC. Asterisks (***, p < 0.001) indicate a significant difference between treatment and control group means ± S.E.M.

Fig. 7. Astrocyte Proliferation in the olfactory lobe glomerular layer following intranasally administered vanadium

Male C57 black mice were intranasally administered 50 µl (182 ug) of vanadium as described in the methods. The vehicle control group was administered de-ionized water. These treated animals were intracardially perfused and the astrocytes in the olfactory lobe were stained for GFAP by immunohistochemistry. A representative 20× pictures of GFAP astroglia in the olfactory lobe of the control and vanadium treated mice is shown. The data is representative of 3 mice per treatment group.