The effect of low dose organic arsenic exposure on inflammatory genes expression in rat’s kidney

Monosodium methylarsonate (MSMA) is an organic arsenical pesticide widely used in agriculture. Exposure to arsenic has been linked with multiple health problems. Inflammatory genes such as interleukin 6 (IL-6) and interleukin 8 (IL-8) play an important role in the pathophysiology of exposure to an acute high dose arsenic-mediated nephrotoxicity, which led to the proximal tubular injury. However, studies focusing on low dose organic arsenic exposure and its adverse effects on kidneys are limited. This study aimed to evaluate the effects of low dose arsenic exposure on the inflammatory genes expression in rats’ kidneys at three different duration intervals; 2 months, four months and six months. Thirty-six male Sprague-Dawley rats were randomly divided into six groups (n=6); a treatment group and its control for each interval. The treatment groups were given daily oral gavage of MSMA at 63.0 mg/kg body weight (BW) which is equivalent to 1/20 LD50 of MSMA. While control groups received distilled water via oral gavage. At the end of study intervals, the kidney tissues were harvested for arsenic level analysis and molecular analysis. The RNA integrity was confirmed with Qiaxcel analysis. The expressions of inflammatory genes were analysed using RT2 SYBR Green qPCR Mastermix. Tissue arsenic concentration was higher in all treated group. Both IL-6 and IL-8 showed a similar pattern of expressions. Organic arsenic down-regulated IL-6 and IL-8 in 2-month (both fold change -1.03) and 6-month groups (fold change -1.36,-1.15). However, in the 4-month group, both IL-6 and IL-8 were up-regulated (both fold change 1.31). Interestingly, these findings suggest that low dose arsenic exposure has shown the anti-inflammatory effect at 2-month and 6-month. However, 4-month paradoxically demonstrated a pro-inflammatory effect consistent with the tissue arsenic levels

Philadelphia chromosome and FISH- negative, cryptic BCR/ABL1 positive acute myeloid leukaemia: A diagnostic dilemma

Introduction: Acute myeloid leukaemia (AML) with BCR/ABL1 is a provisional entity classified in the most recent WHO classification of AML with recurrent genetic abnormalities. This entity can pose diagnostic dilemmas as it can be confused with blastic phase of chronic myeloid leukamia (CML) or mixed phenotype acute leukaemia (MPAL) with BCR-ABL1. We present here a patient with Philadelphia chromosome negative AML and BCR/ABL1 fusion detected by PCR. Case report: A 65-year-old man presented with fever and constitutional symptoms for three weeks. Physical examination revealed a thin man with hepatomegaly and no appreciable splenomegaly. The full blood picture showed bicytopenia with leucoytosis and 58% blasts cells. The bone marrow aspirate was hypercellular, with 48% MPO-positive blasts cells and heterogenous background of granulocytic cells and abundant eosinophils. Cytogenetic analysis showed 46 XY, del (7q) (q22q23) and no BCR/ABL1 was detected using FISH probe BCR/ABL1. However, using multiplex ARMS PCR, both b3a2 and b2a2 fusion genes were detected. This patient is currently on treatment with standard chemotherapy regime and Nilotinib. Discussion: AML with BCR/ABL1 usually have equal distribution of p190 and p210 transcripts and clinically, is associated with no splenomegaly or basophilia and lower bone marrow cellularity. AML with BCR/ABL1 show aberrations that are normally seen in lymphoid pathologies such as deletions of IKZF1 and/or CDKN2A/B genes. IGH and TCR can also show cryptic deletions.1 These events along with the findings of different rearrangement of BCR/ABL1 reflects the genetic heterogeneity of this subtype. In this patient, the presence of two PCR product both representing major transcript (p210), raises suspicion that there are two different breakpoints. The translocation of BCR exon 13 with ABL1 exon2 (leading to b2a2 transcript) and BCR exon 14 with ABL1 exon2 (leading to b3a2 transcript) would normally be seen on FISH.2 Factors that could contribute to non detection on FISH include these novel breakpoints may not be covered by the standard BCR/ABL1 FISH probes and cryptic rearrangement involving multiple chromosomes. Masked Ph positivity in this patient may be due to insertion of ABL1 into BCR region or the translocation of 9;22 is followed by another translocation of both products leading to fairly normal chromosome morphology.3 The clinical relevance of this findings in the patient is for prognosis. In AML with BCR/ABL1, the prognostic significance is dependant on the concurrent genetic abnormalities, and not solely on the presence of BCR/ABL1. Conclusion: Philadelphia chromosome negative BCR/ABL1 positive acute myeloid leukaemia is extremely rare and can pose diagnostic dilemmas. Moving forward, the utilisation of FISH mapping using Bacterial Artificial Chromosomes (BAC) probes, which can cover both minor and major breakpoint and Whole Chromosomes Painting can offer new insights into the formation of masked Ph chromosomes. Direct sequencing is also useful in detecting smaller insertions/deletions that might be otherwise undetected by FISH or conventional karyotyping

Copy number variation of CNVesv27061 analysis among young adults with high blood pressure using optimized droplet digital polymerase chain reaction (ddPCR) method

Several reports and databases on genomic variants have associated variation in DNA sequences (≥ 1kb), or copy number variation (CNV), with susceptibility to common diseases. However, very few reports are found on hypertension and no study has been reported on CNV in prehypertensive and hypertensive young adult Malaysians. In this comparative cross-sectional study, 133 young adults were recruited, comprising of normotensive (45 subjects), prehypertensive (40 subjects) and mild hypertensive (48 subjects) subjects. DNA for CNV determination was extracted from 3 ml of blood samples collected. CNV esv27061 was analysed using optimized droplet digital polymerase chain reaction (ddPCR) method which has enhanced sensitivity and precision. Frequency distribution patterns of CNV among mild hypertensives showed highest peak copy-number-gain (number of copies more than 2) particularly in copy numbers 3 and 5. The prehypertensive subjects exhibited marked increase in copy number 5 when compared with normotensives. All the subjects in this study showed low frequency distribution pattern for copy numbers 2, 6 and 7. This discovery emphasizes the importance of frequency patterns in determining CNV status of prehypertensive and mild hypertensive subjects. Optimization method in this study showed that the detection of CNV esv27061 is possible in our sample population

A Study of Paraoxonase (PON-1) Activity and Concentration in Coronary Artery Disease Patients in Kuantan, Pahang

Introduction: Paraoxonase 1 (PON1) is a high density lipoprotein (HDL) associated enzyme that is known to inhibit oxidative modification of low density lipoprotein (LDL), thus implicated in the pathogenesis of atherosclerosis and coronary artery disease (CAD). It has been suggested that the variability in this enzyme activity is attributed to polymorphism in PON-1 gene. Nevertheless, even within the same genotype, PON-1 activity and concentration has been shown to vary widely between the different individuals. Therefore, recent studies in various populations have emphasized on the importance of measuring the PON1 activity and concentration in assessing the risk of CAD. The data of such study is however scarce in Malaysia. Objective: The aim of this study was to compare the PON-1 activities and concentration between the healthy controls and CAD patients. Methods: A comparative cross sectional study was carried out on 187 CAD patients in Tengku Ampuan Afzan Hospital, Kuantan and 188 healthy controls. Serum samples were analyzed for PON-1 activities towards paraoxon and phenylacetate as well as for HDLcholesterol. PON1 concentration was expressed as PON1 activity per mmol of HDL. Results: Serum PON-1 activities as well as concentration were found to be lower in CAD patients than in the healthy controls but the results were not significant (p > 0.05). Conclusion: Our finding suggested that PON1 activities and concentration were similar between healthy control and CAD patients in Kuantan, Pahang. A multicentre study may be required to confirm our findings in Malaysian population

Assessment of neuroprotective potential of Tualang honey in alzheimer model of rat

Reduction in cerebral blood flow (CBF) due to aging has been associated with neurodegenerative disorders including Alzheimer’s disease and dementia. Experimentally, a condition of chronic cerebral hypoperfusion due to reduced CBF can be induced by permanent bilateral occlusion of common carotid arteries (2-vessel occlusion, 2VO) in rats. Honey is a natural product that has been widely used since long time as a nutrient, for its therapeutic effects in traditional medicine, and recently as an antioxidant. Since oxidative stress leading to neuroinflammation, resulting in neuronal apoptosis and death, is one of the mechanisms which is thought to play a significant role in chronic degenerative neurological disorders, the present study was planned to assess the neuroprotective role of Honey in chronic cerebral hypoperfusion-induced neurodegeneration. After acclimatization, thirty Sprague Dawley rats weighing 200-250 g were equally divided into three groups. Group A – sham control, Group B – 2VO, and Group C – 2VO-H (treated daily with Honey (1.2 g/kg freshly diluted with distilled water, orally by 30 gavage every morning following 2VO). At 10th week, all the rats were euthanized and the hippocampi were isolated. Viable neuronal cell were count in the hippocampal CA-1 region. The results showed damaged, distorted, irregular cells with shrunken cytoplasm and dark pykonotic nuclei in 2VO rats as compared to sham control (p<0.001). Treatment of rats with honey restored the hippocampal cells to their normal structure and revealed the reduced loss of neurons in 2VO+H rats as compared to untreated 2VO rats (p<0.001). This study shows that Malaysian tualang honey might have therapeutic potential for the treatment of chronic cerebral hypoperfusion related neurodegenerative disorders including Alzheimer’s disease

Coinheritance of Hb Adana with Hb constant spring: A case report

Introduction: Compound heterozygosity of non deletional alpha thalassemia often have more severe presentation compared with coinheritance of non deletional alpha thalassemia with deletional alpha thalassemia. The prevalence of non-deletional alpha thalassemia co inheritance, specifically the Hb Adana/Hb Constant Spring in Malaysia is around 0.4%1. Haemoglobin Adana (Hb Adana) arises from point mutation of Codon 59 of either alpha 1 or alpha 2 gene, which will lead to substitution of GlyAsp, and leads to instability of the haemoglobin molecule. (HBA1: c.179G>A or HBA2: c.179G>A)2. Haemoglobin Constant Spring (Hb CS) on the other hand is an abnormal haemoglobin caused by a mutation at the termination codon of α2-globin gene. Hb Adana is most commonly seen in the Malay population whilst Hb CS is seen in the Chinese populations. For both, the carriers are clinically asymptomatic, and, the diagnosis of Hb Adana is challenging as the protein is not detectable on routine haemoglobin analysis. Non deletional alpha thalassemia, in combination with the deletional mutations mostly have mild-to-moderate anaemia. In contrast, patients who were compound heterozygotes non deletional mutations, generally will have more severe anaemia, and much earlier presentation, usually in childhood 3. We present here, an infant with Hb Adana/Hb CS who presented at three months old with severe anaemia. Case report: An eight-month-old female infant presented at three months of age with severe anaemia and hepatosplenomegaly. Hb at presentation was 4.5 g/dL and the blood film showed microcytosis with marked anisopoikilocytosis with prominent basophilic stippling. Capillary electrophoresis performed revealed lowered Hb A (86.9%), raised Hb F (9.2%) and abnormal peak at Zone C (2.4%). Molecular study performed with ARMS PCR revealed co- inheritance of Hb Constant-Spring and Hb Adana, whilst GAP PCR revealed no deletional mutations. Both parents are asymptomatic. Capillary electrophoresis of her father showed small peak at Zone C (0.6%) with heterozygous Hb Constant Spring detected on ARMS PCR. As for the mother, the haemoglobin analysis revealed no abnormality, however, non deletional missense mutation of Codon 59 was detected on ARMS PCR. No deletional mutation was detected on GAP PCR for both parents. This baby is currently needing monthly blood transfusion, with the aim of stem cell transplant after 1 year of age. Discussion: This case illustrates the severity of coinheritance of non deletional alpha thalassemia in an infant. Because the phenotype of patients with coinheritance of Hb Adana with other alpha thalassemia are varied, it can be difficult to predict clinically the prognosis in terms of blood transfusion requirements and growth. The phenotype largely depends on whether HBA 1 or HBA 2 gene is affected. Codon 59-point mutation affecting HBA 2 gene has generally more severe phenotype. Although Hb Adana that was originally described in Turkey affected the HBA 1 gene, Hb Adana in Malaysia and Indonesia by far affected the HBA 2 gene4. Since Malaysia and Indonesia share common cultures and are in the same region, it is not surprising that the same location of the mutation is observed. This is contributed by the diversity of α thalassemia mutations, with the process of natural selection and genetic drift. Conclusion: Coinheritance of non deletional alpha thalassemia should be considered in a multi-ethnic population like Malaysia. There is a need for early recognition of these patients to ensure appropriate monitoring, treatment and family planning can be inculcated

Protective effect of treatment with black cumin oil on spatial cognitive functions of rats that suffered global cerebrovascular hypoperfusion

The fixed oil of black cumin seeds, Nigella sativa L. (NSO), has shown considerable antioxidant and anti-inflammatory activities. Chronic cerebral hypoperfusion has been linked to neurodegenerative disorders including Alzheimer’s disease (AD)and its subsequent cognitive impairment in which oxidative stress and neuroinflammation are the principal culprits. Cerebrovascular hypoperfusion was experimentally achieved by bilateral common carotid arteries occlusion (2VO) in rats. Morris water maze (MWM) test was employed to assess the effects of NSO on spatial cognitive function before and after 2VO intervention. Rats were divided into long-term memory (LTM) and short-term memory (STM) groups, each was further subdivided into 3 subgroups: sham control, untreated 2VO and NSO treated 2VO group. All subgroups were tested with MWM at the tenth postoperative week. Working memory test results for both sham control and NSO treated groups showed significantly lower escape latency time and total distance travelled than untreated 2VO group. Similarly, LTM and STM MWM tests for sham control and NSO treated groups revealed significantly better maze test performance as compared to untreated 2VO group. Sham control and NSO treated 2VO groups demonstrated superior probe memory test performance as compared to untreated 2VO group. The fixed oil of Nigella sativa seeds has demonstrated noticeable spatial cognitive preservation in rats challenged with chronic cerebral hypoperfusion which indicates a promising prospective neuroprotective effect

The effect of tualang honey in chronic exposure of high cholesterol diet in animal model

Introduction: Hyperlipidaemia accompanies chronic renal disease either as a consequence of the renal dysfunction or as part of generalized metabolic derangements. The aim of this study was to determine the effects of tualang honey (TH) on the kidneys of animal model with chronic exposure to high cholesterol diet. Materials and method: Twenty Sprague-Dawley rats were divided into two groups, the high cholesterol diet (12% CD (n= 16) and standard diet (SD) (n=4) and were fed for 12 weeks. After 12 weeks, the rats in the 12% CD group were subsequently divided into four groups. The first group was continued with only 12% CD while the other 3 groups in addition to the 12% CD, they were given TH treatment at different concentrations (1.2, 2.4 and 3.0 g/kg/day) for 4 weeks. Biochemical analysis of lipid profile and renal function were performed at the end of the experiment. The animals were sacrificed and the kidneys were harvested for histological examination. Results: In the 12 weeks HCD group, the serum cholesterol, LDL-c and creatinine levels were significantly higher) compared to that of the SD group. All groups with the tualang honey treatment had significant reduction in the LDL-c, triglyceride and creatinine levels. Histological examination of the kidney tissues of the HCD and HCD+TH groups at 16 weeks revealed segmental mesangial proliferation of some glomeruli with focal mesangial matrix expansion. No areas of periglomerular and peritubular fibrosis were observed. Conclusion: Tualang honey supplementation of animal model with chronic exposure to high cholesterol diet improved the renal function hence suggesting the its renoprotective effect. However, there were no changes seen in the histology of the kidneys . Additionally, tualang honey showed improvement in the LDL-c and triglyceride levels indicating its lipid lowering activities

Preliminary analyses on detection of SYT-SSX fusion-transcripts in Synovial Sarcoma

Introduction: Synovial Sarcoma is a rare cancer and account for 5-10% of adult soft tissue sarcomas. The tumour exhibits unspecified histogenesis composed primarily of spindle cells with variable epithelial components. Despite establishment of some immunohistochemistry staining, making a definitive diagnosis of synovial sarcoma remains a challenging task. This is due to the histo-morphology and immunophenotypes similarities of this tumour to other types of soft tissue sarcoma. Objective: The current study aims to apply a molecular method for detection of SYT-SSX fusion transcript, a common molecular defect (>90% of the cases) in Synovial Sarcoma irrespective of the histologic subtypes. Method: Paraffin-embedded fixed-tissue (PEFT) blocks of 3 confirmed and 15 possible cases of Synovial Sarcoma were retrieved from Department of Pathology, Tengku Ampuan-Afzan Hospital, Kuantan and subjected to RNA purification using the standard spin column protocol. A one step direct RT-PCR was performed using SYT-SSX and PBGD primer sets for detection of SYT-SSX fusion gene and the reference gene PBGD respectively. Results: Our preliminary molecular analyses showed positive SYT-SSX fusion transcript in all 3 confirmed cases and 5 possible cases of synovial sarcoma. Further analysis is still on going for the remaining samples. Conclusion: Molecular detection of SYT-SSX fusion-transcript is useful in establishing the diagnosis of Synovial Sarcoma

Chronic low dose organic arsenic induced liver structural damage

Over the decades, organic arsenic has been thought to be less toxic than inorganic arsenic. Monosodium methylarsonate (MSMA) is a potent organoarsenical herbicide that is still being used in most Asian countries, even though in some countries the used has been restricted. Other organic studies reported the effects mainly on the gastrointestinal system. However, the evidence on the severity of it to the liver is still insufficient. The study objective was to investigate the effect of organic arsenic (MSMA) exposure on hepatocytes and liver sinusoidal endothelial cells (LSEC). Rats were exposed to MSMA at 63.20 mg/kg daily for 6 months duration through oral gavage daily. Control rats received distilled water ad libitum. At the end of the duration, they were sacrificed and underwent liver perfusion for tissue preservation. Liver tissue was prepared for light microscopy, scanning and transmission electron microscopy. Histopathological and ultrastructural comparison between control and treated rats were qualitatively described. Histopathological and ultrastructurally, MSMA has caused necrotic and apoptotic changes of the liver with a reduction of organelles in hepatocytes and capillarization of LSEC. Chronic low dose organic arsenic exposure showed evidence of toxicity to hepatocytes. Interestingly, LSEC demonstrated survival accommodation