Harm J. Heusinkveld

Neurodegenerative and neurological disorders by small inhaled particles.

The worlds population is steadily ageing and as a result, health conditions related to ageing, such as dementia, have become a major public health concern. In 2001, it was estimated that there were almost 5 million Europeans suffering from Alzheimers disease (AD) and this figure has been projected to almost double by 2040. About 40% of people over 85 suffer from AD, and another 10% from Parkinsons disease (PD). The majority of AD and PD cases are of sporadic origin and environmental factors play an important role in the aetiology. Epidemiological research identified airborne particulate matter (PM) as one of the environmental factors potentially involved in AD and PD pathogenesis. Also, cumulating evidence demonstrates that the smallest sizes of the inhalable fraction of ambient particulate matter, also referred to as ultrafine particulate matter or nano-sized particles, are capable of inducing effects beyond the respiratory system. Translocation of very small particles via the olfactory epithelium in the nose or via uptake into the circulation has been demonstrated through experimental rodent studies with engineered nanoparticles. Outdoor air pollution has been linked to several health effects including oxidative stress and neuroinflammation that may ultimately result in neurodegeneration and cognitive impairment. This review aims to evaluate the relationship between exposure to inhaled ambient particles and neurodegeneration.

Hexabromocyclododecane inhibits depolarization-induced increase in intracellular calcium levels and neurotransmitter release in PC12 cells.

Environmental levels of the brominated flame retardant (BFR) hexabromocyclododecane (HBCD) have been increasing. HBCD has been shown to cause adverse effects on learning and behavior in mice, as well as on dopamine uptake in rat synaptosomes and synaptic vesicles. For other BFRs, alterations in the intracellular Ca(2+) homeostasis have been observed. Therefore, the aim of this study was to investigate whether the technical HBCD mixture and individual stereoisomers affect the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a neuroendocrine in vitro model (PC12 cells). [Ca(2+)](i) and vesicular catecholamine release were measured using respectively single-cell Fura-2 imaging and amperometry. Exposure of PC12 cells to the technical HBCD mixture or individual stereoisomers did neither affect basal [Ca(2+)](i), nor the frequency of basal neurotransmitter release. However, exposure to HBCD (0-20 microM) did cause a dose-dependent reduction of a subsequent depolarization-evoked increase in [Ca(2+)](i). This effect was apparent only when HBCD was applied at least 5 min before depolarization (maximum effect after 20 min exposure). The effects of alpha- and beta-HBCD were comparable to that of the technical mixture, whereas the inhibitory effect of gamma-HBCD was larger. Using specific blockers of L-, N- or P/Q-type voltage-gated Ca(2+) channels (VGCCs) it was shown that the inhibitory effect of HBCD is not VGCC-specific. Additionally, the number of cells showing depolarization-evoked neurotransmitter release was markedly reduced following HBCD exposure. Summarizing, HBCD inhibits depolarization-evoked [Ca(2+)](i) and neurotransmitter release. As increasing HBCD levels should be anticipated, these findings justify additional efforts to establish an adequate exposure, hazard and risk assessment.

Bromination Pattern of Hydroxylated Metabolites of BDE-47 Affects Their Potency to Release Calcium from Intracellular Stores in PC12 Cells

Background Brominated flame retardants, including the widely used polybrominated diphenyl ethers (PBDEs), have been detected in humans, raising concern about possible neurotoxicity. Recent research demonstrated that the hydroxylated metabolite 6-OH-BDE-47 increases neurotransmitter release by releasing calcium ions (Ca2+) from intracellular stores at much lower concentrations than its environmentally relevant parent congener BDE-47. Recently, several other hydroxylated BDE-47 metabolites, besides 6-OH-BDE-47, have been detected in human serum and cord blood. Objective and Methods To investigate the neurotoxic potential of other environmentally relevant PBDEs and their metabolites, we examined and compared the acute effects of BDE-47, BDE-49, BDE-99, BDE-100, BDE-153, and several metabolites of BDE-47—6-OH-BDE-47 (and its methoxylated analog 6-MeO-BDE-47), 6′-OH-BDE-49, 5-OH-BDE-47, 3-OH-BDE-47, and 4′-OH-BDE-49—on intracellular Ca2+ concentration ([Ca2+]i), measured using the Ca2+-responsive dye Fura-2 in neuroendocrine pheochromocytoma (PC12) cells. Results In contrast to the parent PBDEs and 6-MeO-BDE-47, all hydroxylated metabolites induced Ca2+ release from intracellular stores, although with different lowest observed effect concentrations (LOECs). The major intracellular Ca2+ sources were either endoplasmic reticulum (ER; 5-OH-BDE-47 and 6′-OH-BDE-49) or both ER and mitochondria (6-OH-BDE-47, 3-OH-BDE-47, and 4′-OH-BDE-49). When investigating fluctuations in [Ca2+]i, which is a more subtle end point, we observed lower LOECs for 6-OH-BDE-47 and 4′-OH-BDE-49, as well as for BDE-47. Conclusions The present findings demonstrate that hydroxylated metabolites of BDE-47 cause disturbance of the [Ca2+]i. Importantly, shielding of the OH group on both sides with bromine atoms and/or the ether bond to the other phenyl ring lowers the potency of hydroxylated PBDE metabolites.

Organochlorine insecticides lindane and dieldrin and their binary mixture disturb calcium homeostasis in dopaminergic PC12 cells

Current hypotheses link long-term environmental exposure of humans to persistent organochlorine (OC) insecticides lindane (HCH) and dieldrin (HEOD) to the development of neurodegenerative disorders, such as Parkinsons disease. Primary adverse neurological effects of these insecticides are directed at inhibition of GABA(A) and glycine receptors, although GABA-independent effects have also been reported. In this paper we describe the effect of dieldrin and a binary mixture of dieldrin and lindane on a critical parameter of neuronal function and survival, i.e., intracellular calcium homeostasis. The intracellular calcium concentration ([Ca(2+)](i)) has been monitored using real-time single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive dye Fura-2. The results demonstrate that nanomolar concentrations of dieldrin time- and concentration-dependently inhibit depolarization-evoked influx of Ca(2+). Co-exposure of PC12 cells to a mixture of dieldrin and lindane revealed an additive inhibition of the depolarization-evoked increase in [Ca(2+)](i), whereas the lindane-induced increase in basal [Ca(2+)](i) is inhibited by dieldrin. The combined findings indicate that dieldrin and binary mixtures of organochlorines affect [Ca(2+)](i) already at concentrations below commonly accepted effect concentrations and close to human internal dose levels. Consequently, current findings illustrate the need to take mixtures of OC insecticides into account in human risk assessment.

Azole Fungicides Disturb Intracellular Ca2+ in an Additive Manner in Dopaminergic PC12 Cells

Humans are exposed to complex mixtures of pesticides and other compounds, mainly via food. Azole fungicides are broad spectrum antifungal compounds used in agriculture and in human and veterinary medicine. The mechanism of antifungal action relies on inhibition of CYP51, resulting in inhibition of fungal cell growth. Known adverse health effects of azole fungicides are mainly linked to CYP inhibition. Additionally, azole fungicide-induced neurotoxicity has been reported, though the underlying mechanism(s) are largely unknown. We therefore investigated the effects of a group of six azole fungicides (imazalil, flusilazole, fluconazole, tebuconazole, triadimefon, and cyproconazole) on cell viability using a combined alamar Blue/CFDA-AM assay and on oxidative stress using a H2-DCFDA fluorescent assay. As calcium plays a pivotal role in neuronal survival and functioning, effects of these six azole fungicides and binary and quaternary mixtures of azole fungicides on the intracellular calcium concentration ([Ca(2+)]i) were investigated using single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive fluorescent dye Fura-2. Only modest changes in cell viability and ROS production were observed. However, five out of six azole fungicides induced a nonspecific inhibition of voltage-gated calcium channels (VGCCs), though with varying potency. Experiments using binary IC20 and quaternary IC10 mixtures indicated that the inhibitory effects on VGCCs are additive. The combined findings demonstrate modulation of intracellular Ca(2+) via inhibition of VGCCs as a novel mode of action of azole fungicides. Furthermore, mixtures of azole fungicides display additivity, illustrating the need to take mixture effects into account in human risk assessment.

Dual actions of lindane (γ-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells.

The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA(A) and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca(2+) concentration ([Ca(2+)](i)). As Ca(2+) triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca(2+) homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca(2+)](i) imaging experiments with fura-2 demonstrated that lindane (≥ 10 μM) rapidly increases basal exocytosis and basal [Ca(2+)](i). Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca(2+) channels (VGCC). On the other hand, lindane (≥ 3 μM) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca(2+)](i) and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca(2+) influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic. These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca(2+)](i) and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca(2+)](i) and exocytosis. This study further underlines the need to consider presynaptic, non-receptor-mediated effects in human risk assessment.

Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels

Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca(2+) concentration ([Ca(2+)](i)) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca(2+)](i), e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca(2+)](i) are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca(2+)](i) in plate reader systems, though the results of such plate reader-based measurements have been questioned. In view of the increasing use of plate reader systems for measurements of [Ca(2+)](i) a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca(2+)](i) is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca(2+)](i). Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca(2+)](i) is associated with caveats and limitations that require further investigation.

In vitro dopaminergic neurotoxicity of pesticides: a link with neurodegeneration?

Around the globe, chemical compounds are used to treat or repel pests and plagues that pose a threat to food and feed production. From epidemiological studies, it is known that there is a link between exposure to certain chemical classes of these so-called pesticides and the prevalence of neurodegenerative disorders such as Parkinsons disease in humans. However, which particular compound(s) account for this link or what underlying mechanisms are involved is still largely unresolved. The degenerative process in Parkinsons disease is largely limited to the dopaminergic neurons in the basal ganglia. Cellular mechanisms that are implicated in parkinsonian neurodegeneration include mitochondrial dysfunction, oxidative stress, disturbance of intracellular calcium homeostasis and endoplasmic reticulum (ER) stress. A major characteristic that distinguishes the dopaminergic neurons in the basal ganglia from other dopaminergic neurons is a particular reliance on intracellular calcium for spontaneous activity. Considering the energy consuming nature of maintenance of the intracellular calcium homeostasis and its involvement in life and death of a neuron, this may explain the specific vulnerability of this neuronal population. Despite a large variation in primary mechanism of action it has been demonstrated that pesticides from different classes disturb intracellular calcium homeostasis, thus interfering with intracellular calcium signalling. This relates to altered dopaminergic signalling, disturbed protein homeostasis and increased oxidative stress. Therefore, effects of (mixtures of) pesticides on the intracellular calcium homeostasis may play a role in the development of Parkinsons disease in humans. Although human exposure to pesticides via e.g. food often occurs in complex mixtures, (human) risk assessment is largely based on the assessment of single compounds. The discovery of common modes of action across different classes of pesticides therefore underpins the urgency of development of new models and approaches in risk assessment.

Comparison of different in vitro cell models for the assessment of pesticide-induced dopaminergic neurotoxicity

Biomedical and (neuro) toxicity research on (neuro) degenerative diseases still relies strongly on animal models. However, the use of laboratory animals is often undesirable for both ethical and technical reasons. Current in vitro research thus largely relies on tumor derived- or immortalized cell lines. Notably, the suitability of cell lines for studying neurodegeneration is determined by their intrinsic properties. We therefore characterized PC12, SH-SY5Y, MES23.5 and N27 cells with respect to the presence of functional membrane ion channels and receptors as well as for the effects of five known neurotoxic pesticides on cytotoxicity, oxidative stress and parameters of intracellular calcium homeostasis using a combined alamar Blue/CFDA assay, a H2DCFDA assay and single cell fluorescent (Fura-2) calcium imaging, respectively. Although all pesticides demonstrated a certain level of functional neurotoxicity in the different cell lines, our results also demonstrate considerable differences in intrinsic properties and pesticide-induced effects between the cell lines. This clearly indicates that care should be taken when interpreting (neuro)toxicity data as the chosen cell model may greatly influence the outcome.

In vitro neurotoxic hazard characterisation of dinitrophenolic herbicides

Dinitrophenolic compounds are powerful toxicants with a long history of use in agriculture and industry. While (high) human exposure levels are not uncommon, in particular for agricultural workers during the spraying season, the neurotoxic mechanism(s) that underlie the human health effects are largely unknown. We therefore investigated the in vitro effects of two dinitrophenolic herbicides (DNOC and dinoseb) on a battery of neurotoxicity endpoints in (dopaminergic) rat PC12 cells. Cell viability, mitochondrial activity, oxidative stress and caspase activation were assessed using fluorescence-based bioassays (CFDA, alamar Blue, H2DCFDA and Ac-DEVD-AMC, respectively), whereas changes in intracellular [Ca(2+)]i were assessed using single-cell fluorescence microscopy with Fura-2AM. The combined results demonstrate that exposure to both DNOC and dinoseb is linked to calcium release from the endoplasmic reticulum and activation of caspase-mediated apoptotic pathways. In subsequent experiments, immunofluorescent labelling with specific antibodies was used to determine changes in intracellular α-synuclein levels, demonstrating that both DNOC and dinoseb increase levels of intracellular α-synuclein. The combined results indicate that in vitro exposure to DNOC and dinoseb activates pathways that are not only involved in acute neurotoxicity but also in long-term effects as seen in neurodegeneration.