This study investigated the neurotoxic effects of rotenone and 6-hyroxydopamine (6 OHDA), two compounds which have been implicated in Parkinson’s disease (PD). PD is a neurodegenerative disorder that results in the impairment of movement. During the disease process, a group of dopamine-containing cells in the brain region called the Substantia Nigra pars compacta (SNc), degenerate. Whilst genetic factors contribute to approximately 5% of PD cases, the causes of the remaining 95% are unknown. What does seem clear is the pivotal role of mitochondrial dysfunction as observed in post-mortem human tissue. Mitochondrial dysfunction leads to energy depletion and the generation of harmful reactive oxygen species (ROS). However, despite the fact that the involvement of mitochondria in the disease process has been well established, the cellular events that lead to, and result from, mitochondrial dysfunction remain poorly understood. Rotenone and 6 OHDA have been implicated in PD for two reasons: (1) both toxins can relatively selectively kill SNc neurons in animal models of PD, and (2) there is evidence for both compounds having a potential causative role in the etiology of the disease in humans. When 6 OHDA is injected into the brain, or rotenone applied systemically, both toxins cause degeneration of SNc neurons. This ability makes them excellent tools for studying mechanisms of PD in animal models. In addition, both toxins inhibit mitochondrial function. Despite extensive use in models of PD, the mechanisms by which each toxin cause cell damage remains elusive. The first part of this study investigated the acute responses of dopaminergic SNc neurons to rotenone exposure (5 nM – 1 µM). The experiments were conducted on brain slices obtained from rats. Electrophysiological recordings (whole-cell patch-clamp technique) were used to detect activation of specific membrane channels as well as cell firing and changes to the membrane potential. In addition, imaging of several fluorescent dyes sensitive to specific cellular events was carried out. In voltage-clamp experiments, acute rotenone (200 nM – 1 µM) application evoked a concentration-dependent outward current which was mediated by tolbutamide-sensitive KATP channels. The current was associated with a drop in cell input resistance (Rm) and, in current-clamp, membrane hyperpolarization and inhibition of spontaneous action potentials. The mechanisms by which rotenone activates KATP channels is controversial, with some studies suggesting activation by ATP depletion and others by elevated reactive oxygen species (ROS). To address this issue, experiments were conducted with high levels of ATP in the pipette solution. Since the rotenone-induced outward current was unaffected by high ATP levels, it was concluded that KATP channel activation was due to oxidative stress. Indeed, the antioxidant Trolox significantly attenuated the current response. Confirmation of elevated ROS production was obtained by recording increased mitochondrial superoxide production, using the fluorescent dye MitoSOX. In addition, rotenone evoked depolarization of mitochondrial membrane potential (ΔΨm). Measurements of intracellular Ca2+ and Na+ were performed using the fluorescent dyes Fura 2 and SBFI, respectively. Rotenone evoked increases to both [Ca2+]i and [Na+]i in a concentration-dependent manner. The rotenone-induced [Ca2+]i rise was unaffected by blocking KATP channels with Cs+. The elevation of [Ca2+]i is particularly important in relation to cell death, since [Ca2+]i overload is known to activate pathways leading to necrosis and apoptosis. There has been growing interest in the synergistic action of rotenone with other toxins/conditions which also enhance [Ca2+]i. This concept was explored in the present study by testing the relationship between the baseline [Ca2+]i level and the rotenone-induced [Ca2+]i increase. Two approaches were taken. Firstly, baseline [Ca2+]i was deliberately raised by activation of voltage-gated calcium channels. When rotenone was applied in the presence of this raised baseline calcium level, the rotenone-induced [Ca2+]i rise was significantly greater. The second approach involved post-hoc analysis of the relationship between the normal cellular variation in baseline [Ca2+]i and the rotenone-induced [Ca2+]i elevation. This analysis also revealed a dependency of the rotenone-induced [Ca2+]i elevation on the baseline calcium level. From this finding, as well as the observation that rotenone evoked ROS production, Transient Receptor Protein subtype M2 (TRPM2) channels were proposed as the likely underlying mechanism. The potentiation of the rotenone-induced [Ca2+]i rise by an elevation in baseline calcium level can be attributed to the calcium-dependence of ROS-sensitive TRPM2 channels, known to respond with increased channel opening to increased [Ca2+]i. Recent findings from our laboratory have confirmed TRPM2 involvement in rotenone toxicity, since blockade of these channels with ACA reduced the rotenone-induced [Ca2+]i rise (K. Chung, unpublished). Imaging using the fluorescent dye propidium iodide (PI) to label cells with compromised membrane integrity was also conducted in acute midbrain slices. SNc neurons were retrograde-labelled with FluoroGold and then exposed to various toxic insults. The detergent Triton-X100 caused an increase in PI labelling, whilst rotenone and high concentrations of glutamate were ineffective over the period of time investigated (up to 40 min). The second part of this study, also conducted on acute rat midbrain slices, investigated the acute responses of SNc neurons to 6 OHDA (0.2 – 2 mM) exposure. Extracellular recordings of action potential firing were conducted on SNc neurons. 6 OHDA evoked rapid inhibition of firing in a similar manner to dopamine (100 µM). In the presence of D2 dopamine receptor blocker sulpiride, the inhibition of firing evoked by 6 OHDA was delayed, and an initial increase of firing was observed. Blockade of the dopamine transporter with nomifensine reduced the 6 OHDA-induced inhibition of firing, and prevented the persistent inhibition of firing after 6 OHDA washout. For comparison, the response to 6 OHDA of non-dopaminergic neurons in the subthalamic nucleus was also studied. In the subthalamic nucleus, 6 OHDA evoked an increase of spontaneous action potential firing. Rapid application of 6 OHDA (using the picospritz application technique) in voltage-clamp recorded SNc neurons evoked an outward current, similar to that observed after dopamine application. In the presence of sulpiride, 6 OHDA induced an inward current, consistent with the initial increase of firing activity observed in extracellular recordings. Microfluorometric experiments with Fura 2, showed that 6 OHDA evokes an increase in [Ca2+]i. Loading cells with the fluorescent dye Lucifer Yellow enabled visualization of 6 OHDA-induced swelling of the cell body and damage to proximal dendrites. Imaging of SNc neurons loaded with dextran-rhodamine revealed 6 OHDA-induced damage of distal dendrites. The last part of the study was performed on organotypic cultures obtained from slices of the ventral midbrain. These cultures were prepared from newborn transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase-promoter. This fluorescent marker enabled easy identification of dopamine-containing cells (including SNc neurons). Only preliminary experiments were carried out using this preparation. GFP-positive neurons did not show the classic membrane hyperpolarization in response to dopamine. For comparison, recordings from GFP-positive SNc neurons in acute slices obtained from age-matched animals did show a typical hyperpolarizing response to dopamine. GFP-neurons from organotypic cultures also lacked the Ih current – another characteristic feature of SNc neurons in vivo or in acute brain slices. In addition, atypical responses to CNQX (blocker of NMDA receptors) and baclofen (blocker of GABAB receptors) application were identified in GFP-positive neurons. These results demonstrate that the culturing process used in this study alters the functional ‘phenotype’ of dopaminergic neurons, a change which needs to be considered in future studies using this preparation. Chronic exposure of organotypic cultures to low concentration of rotenone (50 nM) evoked a delayed increase of PI labelling indicative of cell death, however technical limitations prevented detection of PI co-localization with GFP was observed. In conclusion, this study identified several key aspects of 6 OHDA and rotenone toxicity in SNc neurons. The most significant novel findings include evidence for ROS activation of KATP channels, presumed involvement of TRPM2 channels in rotenone-induced [Ca2+]i rise, and dopamine-analogous effects of 6 OHDA. The controversial role of KATP channels in neuroprotection was addressed. Findings from this study suggest therapies targeting this channel alone would be of little benefit. The proposed involvement of TRPM2 channels in rotenone-induced [Ca2+]i overload in SNc neurons is particularly interesting as it provides a mechanism for synergism between rotenone and other factors that disrupt [Ca2+]i homeostasis.

Tumor necrosis factor (TNF) is upregulated in a number of cardiomyopathies. This thesis investigates TNF in triggering the expression and activation of matrix metalloproteinases (MMPs) in pressure overload cardiac disease, and explores the role of superoxide. Cardiac pressure overload was generated in adult wild-type and TNF-/- mice by transverse aortic constriction. Isolated cardiomyocytes and cardiofibroblasts from neonatal mice ventricles were treated with recombinant TNF (rTNF), and MMP induction and activation were assessed, with and without apocynin (a NADPH-oxidase inhibitor). TNF-/- mice showed less superoxide production and MMP activation, compared to wild-type mice, following pressure overload. rTNF upregulated the production of NADPH-dependent superoxide in cardiomyocytes as early as 1 hour (24 hours in cardiofibroblasts). rTNF also increased the expression of MMP-9 and MMP-12 in cardiomyocytes more than in cardiofibroblasts, and MMP-8 and MMP-13 more in cardiofibroblasts. This induction in both cardiac cell types was concomitant with superoxide production.

The cardiac sodium-calcium exchanger (NCX) is an important membrane protein which regulates cellular calcium necessary for the optimal contractile function of the heart. NCX has become a focal point in ischemic heart disease (IHD) research as evidence suggests that reactive oxygen species (ROS) produced during IHD can cause NCX to malfunction resulting in an intracellular calcium overload leading to cardiac contractile abnormalities. Therefore, I hypothesized that NCX function is mediated by ROS increasing NCX1 activity during cardiac ischemia-reperfusion. To research this hypothesis, I investigated cellular mechanisms which may play a role in NCX dysfunction and also examined methods to correct NCX function. I found that reactive oxygen species directly and irreversibly modify NCX protein, increasing its activity, thereby worsening the calcium overload which is deleterious to cardiac function. I also elucidated the molecular means by which NCX protein modification occurs. Exploring pharmacological means by which to decrease NCX function to relieve the calcium overload and reduce the damage to the heart, I discovered that ranolazine (Ranexa), indicated for the treatment of angina pectoris inhibits NCX activity directly, thereby further reducing the calcium overload-induced injury to the heart. Furthermore, many IHD patients are also co-morbid for type-2 diabetes. These patients are prescribed sulfonylurea (SU) agents which act at the ATP sensitive K+ channel (KATP). One agent such as glibenclamide is known to have cardiotoxic side effects. Therefore, SUs devoid of any cardiac side effects would beneficial. Interestingly, patients possessing the genetic variant E23K-S1369A KATP channel have improved blood glucose levels with the use of the SU gliclazide. Therefore, I determined the functional mechanism by which gliclazide has increased inhibition at the KATP channel. These findings have implications for type-2 diabetes therapy, in which 20% of the type-2 diabetic population carries the KATP channel variant. In summary, the findings presented in this thesis have implications on treatment strategies in the clinical setting, as a NCX inhibitor can be beneficial in IHD and possibly type-2 diabetes. Moreover, a pharmacogenomic approach in treating type-2 diabetes may also provide a positive outcome when considering co-morbid cardiac complications such as atrial fibrillation and heart failure.

The common risk factors for atherosclerosis, such as obesity, high cholesterol levels, sedentary lifestyle, diabetes and high alcohol intake, only explain approximately 50% of cardiovascular disease events. It is thereby important to identify new mechanisms that can stimulate the process of atherosclerosis. During the past decades, a wide range of investigations have demonstrated connections between infections by the respiratory bacterium Chlamydia pneumoniae and atherosclerosis. Earlier studies have focused on the interaction between C. pneumoniae and monocytes/macrophages, T-lymphocytes, smooth muscle cells and endothelial cells, which are present in the atherosclerotic plaque. However, another important player in atherosclerosis and which is also present in the plaques is the platelet. Activation of platelets can stimulate both initiation and progression of atherosclerosis and thrombosis, which is the ultimate endpoint of the disease. The aim of the present thesis was to investigate the capacity of C. pneumoniae to activate platelets and its role in atherosclerosis. The results show that C. pneumoniae at low concentrations binds to platelets and stimulates platelet aggregation, secretion, reactive oxygen species (ROS) production and oxidation of low-density lipoproteins (LDL), and that these effects are mediated by lipopolysaccharide (LPS). Activation of protein kinase C, nitric oxide synthase and 12-lipoxygenase (12-LOX) was required for platelet ROS production, whereas platelet aggregation was dependent on activation of GpIIb/IIIa. Pharmacological studies showed that the C. pneumoniae-induced platelet activation is prevented by inhibitors against 12-LOX, platelet activating factor (PAF) and the purinergic P2Y1 and P2Y12 receptors, but not against cyclooxygenase (COX). These findings were completely opposite to the effects of these inhibitors on collagen-stimulated platelets. We also present data from a clinical study indicating that percutaneous coronary intervention (PCI or balloon dilatation) leads to release of C. pneumoniae into the circulation, which causes platelet activation and LDL oxidation. In conclusion, these data support a role for C. pneumoniae-induced platelet activation in the process of atherosclerosis. Stimulation of platelets by C. pneumoniae leads to release of growth factors and cytokines, oxidation of LDL and platelet aggregation, which are processes that can stimulate both atherosclerosis and thrombosis. Development of novel drugs that prevent C. pneumoniae-platelet interaction by inhibiting 12-LOX and/or PAF, may be important in the future treatment of cardiovascular disease.

Over 500 million people worldwide suffer from disease associated with intestinal inflammation, including gastric cancer, inflammatory bowel disease, h. pylori infections, and numerous viral and bacterial infections. Although potentially effective therapeutics exist for many of these pathologies, delivery challenges thwart their clinical viability. The objective of this work was to develop drug delivery platforms that could target toxic immunomodulatory therapeutics to diseased intestinal tissues. To meet this objective, we developed an oral delivery vehicle for siRNA and an NF-κB inhibiting nanoparticle that reduces drug-resistance. Small interfering RNA (siRNA) represents a promising treatment strategy for numerous gastrointestinal (GI) diseases; however, the oral delivery of siRNA to inflamed intestinal tissues remains a major challenge. In this presentation, we describe a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can orally deliver siRNA to sites of intestinal inflammation, and thus inhibit gene expression in diseased intestinal tissue. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with TNFα-siRNA (TNFα-TKNs) diminish TNFα messenger RNA (mRNA) levels in the colon and protect mice from intestinal inflammation. Activation of nuclear factor-κB (NF-κB) results in the expression of numerous prosurvival genes that block apoptosis, thus mitigating the efficacy of chemotherapeutics. Paradoxically, all conventional therapeutics for cancer activate NF-κB, and in doing so initiate drug resistance. Although adjuvant strategies that block NF-κB activation could potentiate the activity of chemotherapeutics in drug resistant tumors, clinical evidence suggests that current adjuvant strategies also increase apoptosis in non-malignant cells. In this presentation, we present a nanoparticle, formulated from a polymeric NF-κB-inhibiting prodrug, that target the chemotherapeutic irinotecan (CPT-11) to solid tumors, and thus abrogates CPT-11-mediated drug resistance and inhibits tumor growth. In order to maximize the amount of NF-κB inhibitor delivered to tumors, we synthesized a novel polymeric prodrug, termed PCAPE, that releases the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) as its major degradation product. Using a murine model of colitis-associated cancer, we demonstrate that when administered systemically, CPT-11-loaded PCAPE-nanoparticles (CCNPs) are three time more effective than a cocktail of the free drugs at reducing both tumor multiplicity and tumor size.

An online, semi-continuous instrument to measure both total and gas phase atmospheric reactive oxygen species (ROS) and determine the concentration of ROS in the particle phase (ROS(p)) was developed. This instrument was based on a fluorescent probe for quantifying ambient ROS, specifically 2'7'-dichlorodihydrofluorescin, or DCFH probe. This probe was analyzed for sensitivity to a variety of offline and online parameters for efficient use in a field instrument. The ROS(p) instrument measures the peak light intensity at 530 nm to determine ambient ROS concentrations. ROS particles and gases are collected in a mist chamber in a nebulized mist. The instrument alternates measurements of ROS(p+g), or ROS(tot) by means of an inline filter. Fine (PM₂.₅) (ROS(p) is determined by subtraction of the ROS(g) concentration from the ROS(tot), as the ROS(g) signal could not be excluded. This instrument was tested during the summer (May-July) of 2012 at urban and rural sites in the metropolitan Atlanta and surrounding region. Concentrations of ROS(p) determined from this instrument were often below limit of detection. Average concentrations of ROS(p) were found to be 0.25 nmol/m³ in urban Atlanta (Jefferson St. and Georgia Tech), and 0.15 nmol/m³ in Yorkville, a rural site. A side by side comparison of this method with a filter collection method was made in July. The average ROS(p) offline concentrations were 0.15 nmol/m³. These concentrations were comparable to the online average concentrations of 0.21 nmol/m³ for the same period of time. This average and the majority of the measurements comprising it is dominated by the high limit of detection. The ROS instrument as constructed and operated is an efficient way to conduct ROS(p) measurements at the level of a filter study while reducing the labor intensive filter collection and extraction. In order for this instrument to be successful at measuring ambient ROS in the particle phase, the removal of the gas phase from the current sampling scheme is critical as the ROS(g) concentrations are over 90% of the measured ROS. The system as currently operable is best suited for source measurements, including biomass burning plumes or fresh exhaust to capture immediate formation.

<p>Methylglyoxal (MG) is a reactive glucose metabolite and a known causative factor for hypertension and diabetes. Hydrogen sulfide (H₂S), on the other hand, is a gasotransmitter with multifaceted physiological functions, including anti-oxidant and vasodilatory properties. The present study demonstrates that MG and H₂S can interact with and modulate each other's functions. Upon <i>in vitro</i> incubations, we found that MG and H₂S can directly interact to form three possible MG-H₂S adducts. Furthermore, the endogenous production level of MG or H₂S was significantly reduced in a concentration-dependent manner in rat vascular smooth muscle cells (A-10 cells) treated with NaHS, a H₂S donor, or MG, respectively. Indeed, MG-treated A-10 cells exhibited a concentration-dependent down-regulation of the protein and activity level of cystathionine &gamma;-lyase (CSE), the main H₂S-generating enzyme in the vasculature. Moreover, H₂S can induce the inhibition of MG-generated ROS production in a concentration-dependent manner in A-10 cells. In 6-22 week-old CSE knockout male mice (CSE^-/-), mice with lower levels of vascular H₂S, we observed a significant elevation in MG levels in both plasma and renal extracts. Renal triosephosphates were also significantly increased in the 6-22 week-old CSE^-/- mice. To identify the source of the elevated renal MG levels, we found that the activity of fructose-1,6-bisphosphatase (FBPase), the rate-limiting enzyme in gluconeogenesis, was significantly down-regulated, along with lower levels of its product (fructose-6-phosphate) and higher levels of its substrate (fructose-1,6-bisphosphate) in the kidney of 6-22 week-old CSE^-/- mice. We have also observed lower levels of the gluconeogenic regulator, peroxisome proliferator-activated receptor-&gamma; coactivator (PGC)-1&alpha;, and its down-stream targets, FBPase-1 and -2, phosphoenolpyruvate carboxykinase (PEPCK), and estrogen-related receptor (ERR)&alpha; mRNA expression levels in renal extracts from 6-22 week-old CSE^-/- mice. Likewise, FBPase-1 and -2 mRNA levels were also significantly down-regulated in aorta tissues from 14-16 week-old CSE^-/- mice. Administration of 30 and 50 &#x00B5;M NaHS induced a significant increase in FBPase-1 and PGC-1&alpha; in rat A-10 cells. We have also observed a significant up-regulation of PEPCK and ERR&alpha; mRNA expression levels in 50 &#x00B5;M NaHS-treated A-10 cells, further confirming the involvement of H₂S in regulating the rate of gluconeogenesis and MG formation. Overall, this unique study demonstrates the existence of a negative correlation between MG and H₂S in the vasculature. Further elucidation of this cross-talk phenomenon between MG and H₂S could lead to more elaborate and effective therapeutic regimens to combat metabolic syndrome and its related health complications.</p>

Despite extensive preventative efforts, the problem of controlling infections associated with biomedical materials persists. Bacteria tend to colonize on biocompatible materials and form biofilms; thus, novel biomaterials with antibacterial properties are of great interest. In this thesis, titanium dioxide (TiO2)-associated photocatalysis under ultraviolet (UV) irradiation was investigated as a strategy for developing bioactivity and antibacterial properties on biomaterials. Although much of the work was specifically directed towards dental materials, the results presented are applicable to a wide range of biomaterial applications. Most of the experimental work in the thesis was based on a resin-TiO2 nanocomposite that was prepared by adding 20 wt% TiO2 nanoparticles to a resin-based polymer material. Tests showed that the addition of the nanoparticles endowed the adhesive material with photocatalytic activity without affecting the functional bonding strength. Subsequent studies indicated a number of additional beneficial properties associated with the nanocomposite that appear promising for biomaterial applications. For example, irradiation with UV light induced bioactivity on the otherwise non-bioactive nanocomposite; this was indicated by hydroxyapatite formation on the surface following soaking in Dulbecco’s phosphate-buffered saline. Under UV irradiation, the resin-TiO2 nanocomposite provided effective antibacterial action against both planktonic and biofilm bacteria. UV irradiation of the nanocomposite also provided a prolonged antibacterial effect that continued after removal of the UV light source. UV treatment also reduced bacterial adhesion to the resin-TiO2 surface. The mechanisms involved in the antibacterial effects of TiO2 photocatalysis were studied by investigating the specific contributions of the photocatalytic reaction products (the reactive oxygen species) and their disinfection kinetics. Methods of improving the viability analysis of bacteria subjected to photocatalysis were also developed.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that can be modified to oxygenated PAH (oxyPAHs) derivatives. It is well known that oxyPAHs tend to be much more reactive than their parent compounds. Toxicity can be attributed to direct interaction with target molecules or generation of reactive oxygen species (ROS). Metals are another class of contaminant found ubiquitously throughout the environment. Some metals are toxic at levels below the 1:1 ratio predicted by the biotic ligand model and are thought to manifest toxicity through ROS generation. Often metals and PAHs occur as co-contaminants in industrialized environments, yet little is known about their potential co-toxicity or mechanisms of action in mammalian reproductive function. Previously, we described that a PAH, 9, 10-phenanthrenequinone (PHEQ), inhibited LH-stimulated progesterone secretion in dispersed rat corpus luteal (CL) cells (Nykamp et al., 2001). Viability was decreased in CL cells exposed to PHEQ and 1,2-dihydroxy-anthraquinone (1,2-dhATQ), but not their parent compounds phenanthrene (PHE) or anthracene (ANT). Similarly, LH-stimulated progesterone production in CL cells was inhibited by PHEQ and 1,2-dhATQ, but not PHE. Further investigation revealed that PHEQ, but not PHE, ANT nor 1,2-dhATQ generated ROS in CL cells. Viability experiments were repeated using the choriocarcinoma cell line JEG-3 with similar results. Various metals were assessed for their toxicity to both CL and JEG-3 cells. The endpoints used to measure viability were metabolic activity and membrane integrity. In general, metabolic activity was a more sensitive indicator of toxicity than membrane integrity. The order of toxicity for metals in CL cells was Hg2+ > Cd2+ > Zn2+ > Ni2+ > Cu2+ for metabolic activity and Hg2+ ≈ Zn2+ > Cd2+ > Cu2+ > Ni2+ for membrane integrity. Only Hg2+ and Cu2+ were tested in JEG-3 cells. While Cu2+ was non-toxic, EC50s for Hg2+ metabolic activity and membrane integrity were 20 mM and 23 mM, respectively. Experiments were designed to study the mixtures of metals and PAHs on viability, ROS production, and LH-stimulated progesterone production in CL cells. Mixtures of each metal with either PHEQ or 1,2-dhATQ were incubated with CL cells and their effect on metabolic activity and membrane integrity assessed. Generally, most metal/oxyPAH mixtures displayed only additive toxicity. However, mixtures of Cu2+ and PHEQ showed synergistic toxicity to both metabolic activity and membrane integrity. Mixture studies in JEG-3 cells used only combinations of Cu2+ or Hg2+ with PHEQ or 1,2-dhATQ. Similar results to metabolic activity and membrane integrity in CL cells were observed. Mixtures of Cu2+ and PHEQ or 1,2-dhATQ were tested in CL cells for their effect on LH-stimulated progesterone secretion and ROS production. Additive effects were observed in both LH-stimulated progesterone secretion and ROS production for Cu2+/1,2-dhATQ mixtures while synergistic effects for both parameters were seen with Cu2+/PHEQ. Efforts to determine the site of action for mixtures of Cu2+/PHEQ involved adding the cholesterol analogue, 22-OH cholesterol (22-OHC) to CL cells in the absence of LH. Cytochrome P450 side-chain cleavage (CYP450scc) enzyme operates constitutively and the addition of 22-OHC to CL cells resulting in a 5-fold increase in progesterone production without added LH. Kinetic assays with 22-OHC show that while progesterone secretion was inhibited with PHEQ addition alone, a further significant reduction with both Cu2+ and PHEQ was not observed. The use of forskolin, an activator of adenylate cyclase, did not show any significant enhancement of progesterone secretion with the addition of Cu2+/PHEQ compared to PHEQ alone. The potential targets of Cu2+/PHEQ mixture include any step in the steroidogenic cascade from activation of protein kinase A onward with the proteins of the mitochondria, cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein, being the most likely. Differential display polymerase chain reaction (ddPCR) was a molecular approach taken to determine the effect of PHEQ on JEG-3 gene expression. The genes whose expression appeared to be up-regulated with PHEQ exposure were serine protease inhibitor, Alu repeat sequence, heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 3 (eIF3), nucleoporin-like protein, eukaryotic translation elongation factor 1a1 (eEF1 a 1), autophagy-linked FYVE domain (Alfy), spectrin, and proteasome. Apparent down-regulated genes in JEG-3 cells after PHEQ exposure included poly(ADP-ribose) polymerase 10 (PARP10), polyglutamine binding protein-1 (PQBP-1), heterogeneous ribonuclear ribonucleoprotein C (hnRNP C), eukaryotic translation initiation factor 5A (eIF5A), and keratin. In both cell types, oxyPAHs were more toxic than their parent compounds. Metals showed greater toxicity to metabolic activity than to membrane integrity. Of the combinations tested, only PHEQ and Cu2+ exhibited synergistic toxicity. ROS generation was the likely mechanism behind PHEQ/Cu2+ toxicity. Both cell types used represent critical roles in human reproductive health. The proper production of progesterone, a critical hormone for the maintenance of pregnancy in mammals, represents a unique endpoint for the assessment of toxicity. These results illustrate the need to study modified oxyPAHs, metals and metal/oxyPAH mixtures for their potential impact on human reproductive health.

<p>Methylglyoxal (MG) is a reactive glucose metabolite and a known causative factor for hypertension and diabetes. Hydrogen sulfide (H₂S), on the other hand, is a gasotransmitter with multifaceted physiological functions, including anti-oxidant and vasodilatory properties. The present study demonstrates that MG and H₂S can interact with and modulate each other's functions. Upon <i>in vitro</i> incubations, we found that MG and H₂S can directly interact to form three possible MG-H₂S adducts. Furthermore, the endogenous production level of MG or H₂S was significantly reduced in a concentration-dependent manner in rat vascular smooth muscle cells (A-10 cells) treated with NaHS, a H₂S donor, or MG, respectively. Indeed, MG-treated A-10 cells exhibited a concentration-dependent down-regulation of the protein and activity level of cystathionine &gamma;-lyase (CSE), the main H₂S-generating enzyme in the vasculature. Moreover, H₂S can induce the inhibition of MG-generated ROS production in a concentration-dependent manner in A-10 cells. In 6-22 week-old CSE knockout male mice (CSE^-/-), mice with lower levels of vascular H₂S, we observed a significant elevation in MG levels in both plasma and renal extracts. Renal triosephosphates were also significantly increased in the 6-22 week-old CSE^-/- mice. To identify the source of the elevated renal MG levels, we found that the activity of fructose-1,6-bisphosphatase (FBPase), the rate-limiting enzyme in gluconeogenesis, was significantly down-regulated, along with lower levels of its product (fructose-6-phosphate) and higher levels of its substrate (fructose-1,6-bisphosphate) in the kidney of 6-22 week-old CSE^-/- mice. We have also observed lower levels of the gluconeogenic regulator, peroxisome proliferator-activated receptor-&gamma; coactivator (PGC)-1&alpha;, and its down-stream targets, FBPase-1 and -2, phosphoenolpyruvate carboxykinase (PEPCK), and estrogen-related receptor (ERR)&alpha; mRNA expression levels in renal extracts from 6-22 week-old CSE^-/- mice. Likewise, FBPase-1 and -2 mRNA levels were also significantly down-regulated in aorta tissues from 14-16 week-old CSE^-/- mice. Administration of 30 and 50 &#x00B5;M NaHS induced a significant increase in FBPase-1 and PGC-1&alpha; in rat A-10 cells. We have also observed a significant up-regulation of PEPCK and ERR&alpha; mRNA expression levels in 50 &#x00B5;M NaHS-treated A-10 cells, further confirming the involvement of H₂S in regulating the rate of gluconeogenesis and MG formation. Overall, this unique study demonstrates the existence of a negative correlation between MG and H₂S in the vasculature. Further elucidation of this cross-talk phenomenon between MG and H₂S could lead to more elaborate and effective therapeutic regimens to combat metabolic syndrome and its related health complications.</p>