Parkinson's disease (PD) is a neurodegenerative disorder in the elderly resulting in the damage and death of dopaminergic neurons in the brain [1]. Oxidative stress and mitochondrial dysfunction have been implicated as important contributors to neuronal death induced in substantia nigra of patients with PD [2–4]. In particular, increased lipid peroxidation products have been found in PD brains [5]. During the last three decades, epidemiological and toxicological studies provided data that pesticides are potential toxicants for dopamine-producing neurons and contribute to the development of PD [6]. Accordingly, exposure to pesticides has been associated with increased incidence of PD [7, 8]. Mitochondria are targets for a number of environmental compounds including pesticides [6, 7] such as rotenone [9, 10]. Rotenone is highly lipophillic, easily crosses the blood-brain barrier and accumulates in mitochondria where it binds to complex I, inhibits the flow of electrons and results in generation of superoxide [11]. However, the mechanistic links between the oxidative stress, lipid peroxidation, neuronal death, mitochondrial impairments induced by pesticides have not been identified.

Blood cells and circulating lymphocytes have been often used to study the pathogenic mechanisms of neurodegenerative diseases, including PD. Mitochondrial complex deficit [12, 13] and up-regulation of the α-synuclein gene [14] that render these cells susceptible to apoptosis were detected in lymphocytes from PD patients [15, 16]. Recently, we demonstrated that oxidation of a mitochondria-specific phospholipid, cardiolipin (CL), is essential for the execution of apoptosis (release of proapoptotic factors from mitochondria into the cytosol) in primary rat cortical neurons in vitro [17, 18] and in rat brain in vivo [19, 20]. Intermembrane space hemoprotein, cytochrome c (cyt c) has been identified as a catalyst of CL peroxidation [21]. CL is a negatively charged phospholipid with four fatty acid residues [21]. It is found exclusively in the inner-mitochondrial membrane where it accounts for 25% of all phospholipids [22] and plays a significant role in mitochondria bioenergetics [23–26]. Taking into account that lymphocytes express the catecholaminergic system and, similar to neuronal cells, molecular death machinery, including the release of mitochondrial pro-apoptotic factors into the cytosol, leading to the typical morphological and biochemical characteristics of apoptosis [27–29], we suggested that cyt c can be involved in the generation of specifically oxygenated molecular species of CL in circulating lymphocytes exposed to rotenone.

In this paper, by using lipidomics/oxidative lipidomics approaches we identified and characterized molecular species of CL in human peripheral blood lymphocytes, evaluated specific profiles of rotenone-induced CL oxidation products and established their association with the production of H₂O₂, impairments of mitochondrial dysfunction and apoptosis in human lymphocytes exposed to rotenone.

Although oxygenated fatty acids and phospholipids are critical signaling molecules (and/or biomarkers) in several neurological disorders [44], essential information on molecular targets, particularly specific polyunsaturated molecular species of phospholipids undergoing oxidation and leading to mitochondrial dysfunction and their association with neurodegenerative disease such as PD, is lacking. While the general association of oxidative stress with PD has been emphasized in numerous studies (reviewed in [45–48], attempts to link the products of oxidative modification of different biomolecules to specific pathogenetic pathways of PD were not victorious [5, 49]. This may be due, at least in part, to insufficient information on the diversity and structure of oxidized biomolecules generated in mitochondria – the major metabolic candidate as a source of impaired and oxidatively modified PD-associated molecular species. Among those, CLs – unique and functionally essential phospholipids of mitochondria – may be of particular importance [50–52]. In this paper by using LC/MS-based oxidative lipidomics, we characterized all major molecular species of CL and its oxidized molecular species formed in rotenone-associated dysfunctional mitochondria in human peripheral blood lymphocytes.

Changes in the CL content as well as its composition have been shown to be responsible for mitochondrial dysfunction associated with several pathological conditions [53–56]. CL profile in mitochondria can be changed due to: i) loss of CL as a result of alteration in CL synthase activity [2, 31, 57], ii) altered fatty acid composition of CL as a result of disruption of CL remodeling process [58–60] and, iii) CL oxidation due to ROS generation [55]. Generation of ROS [8, 61] and activation of the intrinsic apoptotic cell death pathway [62] have been associated with rotenone-induced neuron degeneration in vitro and in vivo [53–55], dissipation of MMP, release cyt c from mitochondria into the cytosol and apoptosis [63]. Chronic and systemic inhibition of complex I leads to selective degeneration of dopaminergic neurons and produces neuro-pathological features of PD [4, 64].

Having in mind potential detection of mitochondrial phospholipid biomarkers of PD, we examined human peripheral blood lymphocytes that are often used to identify the mechanism leading to development of neurodegenerative diseases such as PD and Alzheimer disease [8, 65, 66]. In fact, increased apoptosis of lymphocytes in patients with PD has been documented [15, 16]. Both extrinsic and intrinsic apoptotic pathways were recognized in lymphocytes of PD patients [16, 67, 68]. Notably, rotenone induced apoptosis in lymphocytes has been linked to its ability to generate ROS (O2−.∕H2O2) leading to mitochondrial damage [10]. Further rotenone-induced inhibition of complex I activity and impairment of electron transport leading to massive production of ROS [69], and possibly protein and lipid peroxidation have been documented in dopaminergic cell line using BODYPI oxidation assays [70]. However, specific features of CL peroxidation and identification of CL oxidation products as essential factors in mitochondrial stages of lymphocyte apoptosis, have not been studied so far.

Here, we demonstrated that exposure of human lymphocytes to rotenone is associated with mitochondria dysfunction, ROS production, development of apoptotic cell death pathway and accumulation of oxygenated species of highly unsaturated CL containing four C_18:2 residues as well as its metabolite mono-lyso-CL. Detailed structural analysis of CL oxidation products revealed unusual features of rotenone-triggered peroxidation: i) predominant peroxidation of TLCL, CL containing four C_18:2 residues ii) preferential accumulation of oxygenated C_18:2 in sn-1 rather than sn-2 position; iii) quantitative abundance of mono-oxygenated species vs species with two and three oxygen functionalities. Further studies will determine whether these specific features are uniquely associated with the rotenone-driven inhibition of respiratory complex I or may be common to other oxidative routes leading to the execution of apoptotic program in lymphocytes.

Our previous work has identified cyt c, an intermembrane space electron carrier, as a catalyst of the reaction during which it binds CL to yield a complex with CL-specific peroxidase activity [34]. The complex generates oxygenated CL species at the early stage of apoptosis in vitro and in vivo [17, 18, 20, 37, 71, 72]. Moreover, cyt c/CL complexes can interact with α-synuclein to form oligomers with high peroxidase activity [52] thus contributing to the formation of Lewis bodies – a morphological hallmark of PD. These results provided direct evidence for previously suggested conversion of cyt c into peroxidase and its possible role in neurodegenerative process [73], including pathogenesis of PD [74, 75]. Notably, while peroxidase activity cyclooxygenase-2 and peroxidation of non-esterified fatty acids have been linked to the pathogenesis of PD [74, 75] the oxidation of esterified lipids has not been yet investigated. Our data indicate that in rotenone treated lymphocytes, cyt c utilizes ROS, particularly hydrogen peroxide, formed during apoptotic cell death, to cause peroxidation of C_18:2-containing CL species in mitochondria (Fig. 9). In addition, rotenone-induced oxidative stress and ROS production can cause activation of Ca²⁺-independent iPLA₂ [76] resulting in the accumulation of CL hydrolysis products such as mono-lyso-CL and oxygenated fatty acids. iPLA₂ has been identified as the major endogenous type of PLA₂ capable of hydrolyzing peroxidized phospholipids in mitochondria [41, 77].

We found that the most predominant molecular species of CL that underwent oxidative modification after exposure of human lymphocytes to rotenone was tetralinoleoyl-CL with four symmetric C_18:2-residues in both sn-1 and sn-2 positions. In addition we were able to detect endogenously formed TLCLox, non-ox-mono-lyso-CL and C_18:2-ox. To determine which of C_18:2 residues was a preferred substrate of rotenone-induced peroxidation, we treated isolated CL fraction with either PLA₁ or PLA₂ – to produce lyso-CLs and release C_18:2 from the respective sn-1 or sn-2 positions. This resulted in liberation of oxygenated C_18:2 species and di-lyso-CLs whereby higher contents of oxygenated C_18:2 was produced by PLA₁ (as compared to PLA₂). Because 99% of total CLs were hydrolyzed and converted into di-lyso-CLs and FAs, we further analyzed oxygenated species of C_18:2. We found that only one out of four C_18:2 in TLCL molecule (in either sn-1 or sn-2 positions) underwent oxidative modification upon rotenone exposure. Assuming that endogenous iPLA₂ cleaves predominantly C_18:2-ox in sn-2 but not non-ox-C_18:2, C_18:2-ox plus non-ox-mono-lyso-CL should be expected hydrolysis products – in line with our observations. Thus not only CL peroxidation products but also mono-lyso-CL in lymphocytes may be used as biomarkers of PD-associated metabolic disturbances – similar to recent finding in patients with Barth syndrome, a disease associated with mitochondrial dysfunction [78].

While this study has been focused on CLs as well as their oxidized and hydrolyzed metabolites, we have also analyzed several other major classes of phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phopshatidylinositol, phosphatidylserine and sphingomyelin. We found that exposure of lymphocytes to rotenone resulted in significant accumulation of several oxygenated molecular species of TLCL as well as its hydrolysis products – mono-lyso-CL and oxygenated free C_18:2. These rotenone-induced changes were CL-specific: neither oxygenated products nor hydrolysis products were detected in other examined classes of phospholipids. Thus no rotenone-induced remodeling of other phospholipids took place under experimental conditions used.

Apoptotic cell death pathway is activated in lymphocytes of PD patients [15, 16]. Therefore, peripheral blood lymphocytes are considered as potential candidate-biomarkers of mitochondrial dysfunction in PD. Given a recently established role of selective peroxidation of a mitochondria-specific phospholipid, CL, in execution of mitochondrial stages of apoptosis, one can assume that detailed analysis of CL peroxidation products may lead to the development of useful biomarkers. It has been reported that micromolar concentrations of rotenone (10–250 μM) induce apoptosis in isolated human lymphocytes [10]. However, specific role of CL peroxidation as an essential factor in mitochondrial stages of lymphocyte apoptosis, has not been yet studied. In the current work, we found that rotenone (at concentrations of 100 and 250 μM) was effective in stimulating selective accumulation of CL oxidation products and induction of apoptosis in isolated human lymphocytes. While the concentrations of rotenone used may seem to be relatively high, one should consider them in the context of its known toxicity and exposure doses. The estimates of rotenone toxicity for humans are commonly based on animal studies. In rat rotenone PD model with administration of pesticide through I.V. route (3–18 mg/kg/day) [79, 80], its concentration in circulation is within micromolar range. Of note, rotenone is usually sold as 1 – 5% formulation that corresponds to approximately its 30–150 mM solution. Thus agricultural workers can be occupationally exposed to relatively high doses of rotenone.

In conclusion, we demonstrated that exposure of human peripheral blood lymphocytes to a pesticide, rotenone, causes time- and dose-dependent selective oxidation of TLCL, accumulation of its hydrolysis products - mono-lyso-CLs - as well as generation of TLCL oxygenated molecular species associated with mitochondrial dysfunction likely through enzymatic cyt c catalyzed reactions triggered early in apoptosis. We believe that characterization of oxidatively modified CL molecular species as well as identification its hydrolysis products are important for better understanding of PD pathogenesis and may lead to the development of new biomarkers of mitochondrial dysfunction associated with PD.