Samuel Njau

Epigenetic mechanisms in metal toxicity.

The true understanding of epigenetics evolved over time as our knowledge on DNA methylation and chromatin modifications and their effects on gene expression increased. The current flurry of research on epigenetics and the increasing documentation of the effects of various environmental factors on DNA methylation, chromatin modification, as well as on the expression of small non-coding RNAs (ncRNAs) have expanded the scope of research on the etiology of various diseases including cancer. The current review briefly discusses various molecular mechanisms of epigenetic regulation of gene expression, and expands the discussion with examples of heavy metal-induced alterations of gene expression and the associated epigenetic changes.

Disposable pipette extraction for gas chromatographic determination of codeine, morphine, and 6‐monoacetylmorphine in vitreous humor

The availability of a sensitive and rapid analytical method for the determination of opiates, and other substances of forensic interest, in a variety of biological specimens is of utmost importance to forensic laboratories. Solid-phase extraction is very popular in the pre-treatment of forensic samples. Nevertheless, a new approach, disposable pipette extraction (DPX), is gaining increasing interest in sample preparation. DPX has already been applied to the analysis of drugs of abuse in common biological matrices, such as urine and blood, but has not yet been evaluated on alternative biological samples, such as vitreous humor. The objective of this study was to evaluate the applicability of DPX on the analysis of opiates in vitreous humor. The currently developed method is fast, reliable, and easy to perform. The sensitivity, precision, and accuracy are satisfactory. Recoveries obtained are within the range of 72-91%, whereas the sample volume of vitreous humor required is only 100 μL.

Drugs of Abuse: Epigenetic Mechanisms in Toxicity and Addiction

The abuse of substances such as ethanol, cocaine, amphetamines and heroin is associated with toxic effects on almost every system of the organism. Furthermore, the transition from occasional-recreational use to chronic abuse and addiction is a serious psychiatric disorder with only few chances for effective and definitive treatment since most individuals relapse, even after long periods of abstinence. It is therefore of utmost importance to elucidate the mechanisms by which these substances exert their toxicity and mediate addiction, in order to develop new, efficient therapeutic strategies with a long-term outcome, which are currently lacking. We already know that in a great number of these mechanisms, altered gene function is involved. But, with the new field of epigenetics, there is increasing evidence that changes in the epigenome are responsible for the altered gene function. The advances in the field of epigenetics towards elucidation of the mechanisms underlying toxicity and addiction for ethanol, cocaine, amphetamines and heroin are currently presented and discussed in this review.

Glutamate Receptors in Human Caudate Nucleus in Normal Aging and Alzheimer’s Disease

Neostriatum is one of the brain areas that are not primarily affected in Alzheimers disease, according to classic regard of the disease. However, recent data emphasize the involvement of neostriatum, especially the head of the caudate nucleus, in the emergence of characteristic symptoms of the disease. Glutamatergic neurotransmission is a key component of striatal pathways. The present study is focused on glutamate receptors of striatal neurons on human caudate nucleus in normal aging and Alzheimers disease. Immunohistochemical studies were carried out for N-methyl-D-aspartate receptor subunit 1 (NMDAR1), α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit 2 (GluR2) and metabotropic glutamate receptor 5 (mGluR5). Ionotropic receptors (NMDAR1 and GluR2) were found to be expressed by 82% - 93% of striatal neurons with no significant alterations in aging and Alzheimers disease. On the other hand, metabotropic receptor mGluR5 was found to be expressed by just 40% of striatal neurons in young individuals, with significant intensity variations among the neurons. This percent was increased in elderly individuals and Alzheimers disease patients to 80% and 92% of striatal neurons, respectively. The up-regulation of mGluR5 both in normal aging and Alzheimers disease is possibly associated with reorganization of neuronal connections, indicates the complexity of this receptor function and renders quite unpredictable the intervention and treatment of dementia with mGluR5 inhibitors or modulators.

Alterations in serum MMP and TIMP concentrations following chronic heroin abuse

Abstract Context: Although opiate abuse is known to affect matrix metalloproteinases (MMPs), data on these enzymes and their tissue inhibitors in heroin addicts are scarce. Objective: In the present study, we determined serum concentrations of MMP-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in heroin users, and compared them with healthy individuals. We evaluated whether 21 d of abstinence are adequate to reverse the effect of opiates and we compared seropositive with seronegative, for anti-HCV antibodies, heroin users. Materials and methods: Twenty-six heroin-dependent male volunteers and an equal number of healthy individuals participated in this study. ELISA was used to assess the serum levels of MMP-2, MMP-9, TIMP-1 and TIMP-2. Heroin users were assessed both upon admission and upon completion of a 21-d detoxification program. Results: Serum TIMP-1 concentrations were significantly lower and the ratios MMP-2/TIMP-1, MMP-9/TIMP-1 and MMP-2/TIMP-2 were significantly higher in heroin users compared to healthy individuals. Heroin users who were seropositive had lower MMP concentrations, as well as lower MMP/TIMP ratios, compared to those who were seronegative. Discussion: Our results showed that in heroin-addicted individuals, and especially those who are positive for anti-HCV antibodies, the balance between MMPs and TIMPs in serum is disrupted and this disruption cannot be restored within 21 d of abstinence. Conclusion: Chronic heroin abuse disrupts the balance between MMPs and TIMPs in serum and this effect is not reversible within 21 d of abstinence.

A simple HPLC method for the simultaneous determination of two selective serotonin reuptake inhibitors and two serotonin–norepinephrine reuptake inhibitors in hair, nail clippings, and cerebrospinal fluid

A novel and simple high-performance liquid chromatography method has been developed for the simultaneous determination of two selective serotonin reuptake inhibitors (fluoxetine and paroxetine) and two serotonin-norepinephrine reuptake inhibitors (venlafaxine and duloxetine) in alternative samples of toxicological interest such as hair, nail clippings, and cerebrospinal fluid (CSF). The separation was achieved on a Hichrom Kromasil 100-5C(18) (250 × 4.6 mm) 5 μm column by using ammonium acetate (0.05 M)-acetonitrile (59:41% v/v) as the mobile phase, delivered isocratically at a flow rate of 1.3 mL/min, within ca. 10 min. Ultraviolet detection at 235 nm was used for monitoring the eluting analytes. Validation was performed in terms of linearity, selectivity, accuracy, precision, and stability. Correlation coefficients were greater than 0.9954. The limits of quantitation ranged between 0.3 and 2.1 ng/μL for all analytes in the liquid matrix (CSF), while the respective values were in the range of 0.3-3.6 ng/mg for solid matrices (hair and nail clippings), with an injection volume of 20 μL. Repeatability and intermediate precision (relative standard deviation, RSD%) were less than 16.6%. The method was successfully applied to actual hair and nail samples from a patient under fluoxetine treatment.

Neuropathological findings in Dandy Walker Variant

Objective: Dandy-Walker malformation is a congenital malformation that is characterized by hypoplasia of the cerebellar vermis, cystic dilatation of the fourth ventricle and enlargement of the posterior fossa. The aim of the study is to describe the morphological and morphometric alterations of neurons (in various regions of the brain) in a case of Dandy-Walker Variant. Methods: By Golgi method and routine techniques, the alterations in the brain of a 23 years old case of Dandy-Walker Variant who died accidentally were studied. Furthermore, a detailed morphometric analysis of neuronal parameters and a statistical comparison with an age-matched control were applied. Results: In the cerebellum the morphological study, apart from gliosis, revealed reduction of the size of cell body and poverty of dendritic arborization with loss of dendritic branches and spines. In the cerebral cortex and the hippocampus the most prominent findings were the tortuous configuration of the apical dendrites of the pyramidal neurons, the focal swellings of the axons and the dendrites and significant gliosis. Conclusion: Although the gross anatomical examination of the brain demonstrated only mild neurodevelopmental anomalies (except the key features of the syndrome), the microscopic examination revealed significant morphological alterations of neurons and dendrites.

Evaluation of prooxidant-antioxidant balance in chronic heroin users in a single assay: an identification criterion for antioxidant supplementation.

Background: Opiate abuse has been linked to oxidative stress, through the separate evaluation of oxidants and antioxidants. Objectives: To determine prooxidant–antioxidant balance (PAB) in chronic heroin users in a single assay, easily applied in a clinical setting. Specifically, to examine whether PAB values correlate with the duration of abuse or with the presence of anti-HCV antibodies. Methods: Sixty-four chronic heroin users - 34 cases and 30 controls - participated in this study. PAB was determined by an Enzyme-linked immunosorbent assay (ELISA) method, developed by members of the study group. Results: In heroin users, oxidative balance was disrupted in favor of prooxidants. There was no correlation of PAB values with the duration of abuse or with the presence of anti-Hepatitis C virus (HCV) antibodies. Conclusions: Chronic heroin users can benefit from an antioxidant therapy, and the method currently presented can be used as an identification criterion.

Isoprostane as a Marker of Oxidative Stress in Chronic Heroin Users: Correlation with Duration of Heroin Use or Concomitant Hepatitis C Infection

Background: Although chronic heroin abuse has been extensively linked to oxidative stress, and while plasma 15-F2t-IsoP is considered a good indicator of oxidative stress, there remain few references in the literature about the plasma concentration of this marker in heroin dependent subjects. Objectives: To determine plasma 15-F2t-IsoP, as a marker of oxidative stress, in chronic heroin users, and to examine whether the values of this marker correlate with the duration of heroin use or with the presence of anti-HCV antibodies. Methods: Forty-two chronic heroin users and twenty two healthy control subjects were recruited for this study. An enzyme-immunoassay method was used for the determination of 15-F2t-IsoP in plasma. Results: Plasma 15-F2t-IsoP values were significantly higher in chronic heroin users compared to healthy controls. No correlation was found between the values of plasma 15-F2t-IsoP and the duration of heroin use. Heroin dependent subjects positive for anti-HCV antibodies had significantly lower values of plasma 15-F2t-IsoP as compared to those without a history of HCV infection. Conclusions: The elevated plasma 15-F2t-IsoP values in heroin dependent subjects, compared to healthy individuals, indicate a shift of the balance between oxidants and antioxidants towards the former and suggest that heroin dependent subjects could benefit from an antioxidant therapy.

The combination of silver techniques for studying the pathology of Alzheimer's disease.

Alzheimers diseaseis a progressive neurodegenerative disorder, which implicates the whole central nervous system. The hallmarks of the disease are the development of neuritic plaques and neurofibrillary tangles, the accumulation of β-amyloid in the cytoplasm of the neurons (soluble β-amyloid oligomers) and the neuropile space (insoluble amyloidal fibrils), the neuronal loss, and the devastating synaptic alterations. Despite the fact that for the identification of the plaques and tangles and for the detection of the amyloid deposits and the neuronal loss, there are specific techniques even in light microscopy, synaptic pathology can be studied only with electron microscopy or indirectly with immunohistochemistry, because several alterations in the density of proteins located in synaptic junction (drebrin, synapsin, synaptophisin) may be occurring. Thus, this article presents original Nauta method impregnating degenerating axons as well as axonic terminals in post-mortem material derived from patients suffering from Alzheimers disease. Furthermore, the article proposes its application in combination with Golgi method and Gallyas technique for a spheroid view of the neuronal degeneration and synaptic pathology in the study of any brain region in Alzheimers disease.