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Background: The study of gene-environment interactions enables us to further understand the pathogenesis of asthma and inflammation. The TNF-α gene has been associated with airway pathology in asthma but there is limited information in relation to pollutant exposure and the TNF-α 308G/A polymorphism. Objective: To determine the risk conferred by the TNF-α 308(G/A) polymorphism on respiratory outcome and to evaluate whether the association between exposure to ambient air pollutants such as SO2, NO2, NO, and PM10 and variation in lung function measures is modified by genotype. Methods: The sample comprised 129 African children (between 9-11 years old). A questionnaire based on guidelines from the British Medical Research Council and the American Thoracic Society was administered to all caregivers to evaluate the prevalence of respiratory symptoms. Atopy was evaluated by skin prick testing. Bihourly measures of lung function (spirometry) were collected at school five days per week over three week periods in each of four seasons (2004-2005) using digital hand-held devices. During each of the four intensive 3-week phases, gaseous air pollutant concentrations were monitored continuously. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-PFLP) analysis was used to detect the TNF-α 308 genotype and plasma TNF-α levels were measured using the human TNF-α Max Standard™ Enzyme-linked immuno-absorbent assay (ELISA) kit. Results: The TNF-α variant A allele was common among this sample of African children (40% with an allelic frequency of 0.24). There was no significant association with the TNF-α G/A polymorphism and any respiratory linked phenotype, nor cytokine levels. However, when exposure to pollutants were analyzed with genotypic and phenotypic data, we found relatively modest interaction effects for the TNF-α 308 genotype. GEE models showed that children with the TNF-α 308 A allele had increased deterioration of lung function post pollution exposure to SO2 [β=2.62, CI:0.51-4.71, p=0.02 and pint=0.03] and NO [β=3.28, CI:0.68-5.89, p=0.01, pint=0.03]. Conclusion: The TNF-α 308 (G/A) polymorphism may be associated with increased pollutant-associated effects on FEV1 intraday variability for both SO2 and NO. The A allele may increase susceptibility to the adverse effects of air pollutants. Key words: TNF-308α G/A, respiratory phenotypes, air pollution, asthma, biomarker DOI 10.12932/AP0677

In humans and other multicellular organisms that have an extended lifespan, the leading causes of death are atherosclerotic cardiovascular disease and cancer. Experimental and clinical evidence indicates that these age-related disorders are linked through dysregulation of telomere homeostasis. Telomeres are DNA protein structures located at the terminal end of chromosomes and shorten with each cycle of cell replication, thereby reflecting the biological age of an organism. Critically shortened telomeres provoke cellular senescence and apoptosis, impairing the function and viability of a cell. The endothelial cells within atherosclerotic plaques have been shown to display features of cellular senescence. Studies have consistently demonstrated an association between shortened telomere length and coronary artery disease (CAD). Several of the CAD risk factors and particularly type 2 diabetes are linked to telomere shortening and cellular senescence. Our interest in telomere biology was prompted by the high incidence of premature CAD and diabetes in a subset of our population, and the hypothesis that these conditions are premature-ageing syndromes. The assessment of telomere length may serve as a better predictor of cardiovascular risk and mortality than currently available risk markers, and anti-senescence therapy targeting the telomere complex is emerging as a new strategy in the treatment of atherosclerosis. We review the evidence linking telomere biology to atherosclerosis and discuss methods to preserve telomere length.

Background Currently used 3rd generation rapid HIV-tests in resource-limited settings do not detect acute HIV-infections (AHI). They are known to detect HIV-infections after or late in the "window period". Detecting incident-HIV infections early in pregnancy increases opportunities for initiating antiretroviral prophylaxis to prevent mother-to-child transmission of HIV. Objectives We evaluated the Determine ® HIV1/2 Ag/Ab Combo Rapid Test (Combo RT), a 4th generation test against two 3rd generation tests (SENSA-HIV1/2/0 Tri-line, SD-Bioline) for early detection of HIV-infection in pregnancy. Study design In a cohort study, plasma samples from 32 pregnant women who seroconverted at a subsequent antenatal visit (incident-infection), samples from 189 women who tested HIV positive at baseline (established-infections) and samples from 32 women remaining HIV-seronegative at a subsequent antenatal visit were tested with 3rd generation (antibody detection only) and 4th generation (antibody/antigen detection) rapid HIV tests. The HIV-1 NucliSENSEasyQ ® v2.0 PCR test was used to quantify HIV-viral copies in women with incident HIV-infections. Results Eighteen of 32 (56.3%) women (incident-infections) had detectable viral copies (baseline); 16 (88.9%) were antibody reactive with the Combo RT. None of the 32 specimens were reactive on the antigen component of the Combo RT. The sensitivity and specificity of the Combo RT in detecting HIV infections prior to seroconversion is 59.4% (95%CI 40.6–76.3) and 96.9% (95%CI 83.8–99.9) respectively. The Combo RT detected 94.0% of all HIV-infections if used as a screening test (baseline) compared to 85.5% detected by 3rd generation tests. Conclusions The Combo RT does not identify AHI but is superior to 3rd generation tests in detecting HIV antibody responses.

In a mycological study, a total of 95 human food samples were investigated to evaluate the incidence of fungal contamination in Cameroon by conventional identification method and partly confirmed by DNA sequencing. The isolated fungal spp. were further studied to determine their toxigenic potentials. The investigation revealed the predominance of Aspergillus and Penicillium with 96% of samples contaminated with at least one species of these fungi, whereas the incidence of co-contamination of samples was 85%. Aspergillus flavus and Aspergillus parasiticus (Flavi section) were the most predominant species contaminating mainly maize and peanuts. In addition, P. crustosum and P. polonicum were the most common contaminants belonging to the genus Penicillium. On the other hand, A. ochraceus (Circumdati section) registered a low incidence rate of 5%, including other members of the Aspergillus group. Other members of the genera Rhizopus and Alternaria spp. were also registered in the study. A majority of fungal strains of A. ochraceus, A. parasiticus, P. crustosum and P. polonicum isolated were toxigenic, producing the mycotoxins tested for, while none was detected in cultures of A. fumigatus. The high incidence rate of fungi contamination coupled with their potentials in producing mycotoxins gives a strong indication that the samples tested may likely be contaminated with various mycotoxins. There is need for further study to assess the incidence of mycotoxins contamination in similar food samples.

T-helper (Th) 17 cells are a pro-inflammatory subset of CD4(+) effector T-cells critical in mucosal immunity. Imbalances in Th17 cell proportion have been implicated in the pathogenesis of several diseases; however, this has not been adequately explored in tuberculosis (TB) and human immunodeficiency virus (HIV) co-infection. Since Th17 cells are predominantly mucosally associated, we assessed Th17 proportion and associated microenvironment in pleural effusions from patients co-infected with TB/HIV. Our results show that TB(+)HIV(+) pleurisy results in significantly reduced frequency of CD4(+)IL-17(+)RORC(+)STAT3(+) Th17 cells compared to TB(-)HIV(-)ex vivo (p = 0.0054) and was confirmed in conditioned media studies in vitro (p = 0.0001). This was not associated with alterations in Th17 polarising cytokines IL-6, IL-21 and IL-23 or changes in Th17 signature cytokines IL-17A and F. However, the mRNA expression of Th17 signalling molecules, IL-6 (p = 0.0022), IL-6R (p = 0.0247), IL-1β (p = 0.0022) and signal transducer and activator (STAT) 3 (p = 0.0022) were significantly upregulated. Notably, TB(+)HIV(+) pleural fluid contained significantly higher concentrations of IL-1β (p = 0.0008), IL-22 (p = 0.0115), IL-31 (p = 0.0210), TNF-α (p = 0.0251) and IFN-γ (p = 0.0026) than TB(-)HIV(-) pleural fluid ex vivo. Taken together, this suggests a reduced portion of Th17 lymphocytes in TB/HIV pleurisy is independent of locally mediated cytokine polarisation.

Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc.

Hyperglycemia exacerbates the production of mitochondrial reactive oxygen species and this contributes to a variety of pathological conditions. Sirtuin 3 (SIRT3) has been shown to play a role in decreasing oxidative stress and improving disease outcomes by regulating antioxidant defense. Our understanding of molecular events during oxidative stress under chronic hyperglycemia in the liver is limited. We postulated that SIRT3 may play a role in antioxidant defense under hyperglycemic conditions in HepG2 cells.Cell viability was determined in HepG2 and human embryonic kidney (HEK) 293 cells cultured in the presence of 5.5 mM glucose (control), 19.9 mM mannitol (osmotic control), 10 mM glucose and 30 mM glucose (hyperglycemic), and nicotinamide (NAM) (10 mM) at 24-hr and 72-hr time points. SIRT3, peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and cyclic adenosoine monophosphate (cAMP) response element binding protein (pCREB) protein expression were measured via western blotting. Mitochondrial antioxidant enzymes glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), and uncoupling protein 2 (UCP2) and mitochondrial DNA (mtDNA) repair enzyme 8-oxoguanine glycosylase (OGG1) were evaluated via quantitative PCR (qPCR).Increased cell viability and protein expression of SIRT3, pCREB, and PGC-1α were observed under hyperglycemic conditions at 24 hr. These were further elevated at the 72-hr time point. We also observed higher fold changes of SIRT3, GPx1, SOD2, UCP2, and OGG1 in the treated groups. Treatment with NAM decreased protein and gene expression of SIRT3, pCREB, PGC-1α, GPx1, SOD2, UCP2, and OGG1 at both time points in the hyperglycemic groups.Our data may allude to the relationship that has been established between SIRT3 and PGC-1α with regard to increased antioxidant defense during oxidative stress. This suggests that SIRT3 may play a role in increasing antioxidant defense and conferring resistance to oxidative stress-induced damage under hyperglycemic conditions in the human hepatoma cell line.

To determine the effect of petrol exposure on DNA integrity in peripheral blood lymphocytes among petrol attendants and a non-exposed comparison population. This cross-sectional study included 101 fuel station employees and 50 office-based non-exposed workers in Durban, South Africa. Participants were interviewed using a validated questionnaire. Genomic DNA was extracted from peripheral lymphocytes for the benzo(a)pyrene diol epoxide (BPDE)-DNA adduct assay (ELISA), and DNA damage was determined using the comet assay and reported as percentage tail DNA. The exposed (n = 101) and non-exposed participants (n = 50) varied with regard to age, housing, smoking, and proximity to industry and petrol stations. Among the exposed, the mean duration of employment in the fuel industry was 5.8 years (SD = 4.6), and among those pumping fuel (n = 75), the mean metric tons of petrol pumped in the past 12 months per worker was 199.2 (SD = 88.9). The mean percentage tail DNA varied significantly between exposed and non-exposed groups: 23.8 % (SD = 13.3) and 8.1 % (SD = 1.8) (p

Recent research has demonstrated that lymphocyte apoptosis sensitivity appears to be related to training status and exercise intensity. This work investigated the effect of prolonged, submaximal treadmill running on percentage (%) apoptosis, % necrosis and DNA strand breaks in lymphocytes and related these to changes in total lymphocyte and blood cortisol concentrations in well-trained runners. Venous blood samples (n = 14) were taken immediately before (PRE), immediately after (IPE) and 3 h after (3PE) 2.5 h of treadmill running at 75% of VO2 max from eight well-trained male endurance athletes (age 34.2 +/- 2.44 years) and analysed for cellular content and serum cortisol concentrations. Lymphocytes were isolated from whole blood and % apoptotic and necrotic cell were detected by flow cytometry using Annexin V-FITC and propidium iodide uptake. DNA strand breaks were measured by single-cell gel electrophoresis. Despite a significant (P0.001) exercise-induced increase in mean serum cortisol concentrations and reduction in lymphocyte counts, the mean % Annexin-V positive cells (13.3 +/- 6.78 in PRE, 11.3 +/- 5.51 in IPE and 12.8 +/- 6.75 in 3PE samples) were not significantly different at the three time-points (P0.05). Mean DNA strand breaks in the lymphocytes also did not change significantly (P0.05) rising from 25.7 +/- 2.16 to 26.9 +/- 1.89 and 27.1 +/- 1.38 microm in IPE and 3PE samples, respectively. The exercise-induced changes in total blood lymphocyte counts and cortisol concentrations did not result in a significant change in % apoptotic lymphocytes or DNA strand breaks in the endurance-trained athletes during this prolonged, submaximal exercise.

Atorvastatin is used to control cholesterol and lipid levels in hyperlipidaemic and hypercholesterolaemic patients. Myopathy and hepatotoxicity, however, have been reported as side effects in a small percentage of statin users. This study aimed to investigate the cytotoxicity and the effect of atorvastatin on microRNA expression in HepG2 cells. The methylthiazol tetrazolium assay was used to assess hepatocyte viability and at 20 μM atorvastatin (24 h) treatment were 82 ± 1.5% viable (P = 0.0002). Levels of intracellular ATP in cells treated with 20 μM atorvastatin were reduced by 1.25-fold, P = 0.002. Cytotoxicity, measured by the release of intracellular lactate dehydrogenase, was increased from 0.95 ± 0.29 units in control cells to 1.12 ± 0.02 units (P = 0.002) in atorvastatin treated cells. A panel of 84-miRNA species was used to evaluate the effect of atorvastatin on miRNA expression. MiR-124a was significantly up-regulated by atorvastatin (12.94-fold). A significant decrease in GAMT expression (3.54-fold) was observed in atorvastatin treated cells following quantitative PCR analysis. In addition, western blotting data showed GAMT protein levels were significantly lower than the controls (3.02-fold) and analysis of creatine levels in treated cells showed a significant decrease in the atorvastatin treated culture supernatant compared to control culture supernatant (32.33 ± 3.51 μM/l vs. 59.67 ± 1.52μM/l, P = 0.0056). This is the first study to show that atorvastatin up-regulates miR-124a levels and consequently modulates GAMT expression in hepatocytes. J. Cell. Biochem. 116: 2620–2627, 2015. © 2015 Wiley Periodicals, Inc.

Lon protease, an ATP dependent mitochondrial protease, is important in mitochondrial protein maintenance. Disruption of protein homeostasis and mitochondrial dysfunction is associated with lipodystrophy, metabolic syndrome and accelerated aging, and are commonly observed in patients on long term antiretroviral therapy. Sirtuin 3 (SIRT3) is a post-translational regulator of Lon and regulates antioxidant response. We previously showed the nucleoside analogues (NRTIs), Zidovudine (AZT; 7.1 μM), Stavudine (d4T; 4 μM), and Tenofovir (TFV; 1.2 μM) induced oxidative stress and mitochondrial dysfunction in human hepatoma (HepG2) cells at 24 h (h) and 120 h. We conducted a mitochondrial proteomic assessment of homeostasis in the same model, using the same NRTIs. Protein expression of Lon, SIRT3, heat shock protein (HSP) 60, phospho-eukaryotic translation initiation factor 2α (p-eIF2α; Ser51) and phospho-c-jun N-terminal kinase (p-JNK; Thr183/Tyr185) were quantified by western blots. The data showed all stress responses were significantly increased in HepG2 cells by all antiretroviral drugs at 24 h (p

Summary The increased accessibility of antiretroviral therapy continues to positively drive the reduction in viral load and survival of patients despite the attendant reproductive toxicities. We propose that testicular damage caused by highly active antiretroviral therapy (HAART) can be attenuated by antioxidant treatment by investigating the testicular histomorphologic and stereological effects of antiretroviral drugs and its interaction with antioxidants using an experimental animal model. Sprague–Dawley rats were divided into seven groups of six rats per group (A, B… G) using simple random sampling and treated orally with 0.9% normal saline as placebo, a HAART cocktail of stavudine, lamivudine and nevirapine using the adjusted human therapeutic doses of 200, 600 and 350–400 mg/day, respectively, and antioxidants ascorbic acid (vitamin C) and I.M α-tocopherol (vitamin E). Animals were killed after 4 weeks and testicular tissue harvested and processed for light microscopy and stereological evaluations. The results were interpreted by a Veterinary pathologist blinded to the study. No animal died during the experimental period. The histopathological assessment of the testis of animals treated with placebo, ascorbic acid alone and α-tocopherol alone as well as vitamin E + HAART displayed normal testicular microanatomy. Groups treated with HAART alone, HAART + vitamin C + vitamin E and vitamins C + HAART showed extensive seminiferous tubular atrophy, necrosis and hypocellularity in the histoarchitectural patterns. While testicular cross-sectional area of seminiferous tubules remained unaffected by HAART, epithelial heights significantly decreased (p

Aflatoxin B1 (AFB1 ) is a carcinogenic mycotoxin found in feeds and in airborne grain dusts. Aflatoxin B1 requires biotransformation to the AFB1-8,9 epoxide (AFBO) by a bioactivation system and subsequent covalent binding to DNA or proteins, to exert its carcinogenic potential. The lung contains cytochrome P450, prostaglandin-H-synthase, lipoxygenase, epoxide hydrolase and other bioactivation enzymes, and is thus a potential target for the effects of AFB1 via the routes of inhalation and ingestion. The A549 human epithelioid lung cell line and the methylthiazol tetrazolium (MTT) bioassay were used to investigate the cytotoxicity of AFB1 and its chemically synthesised epoxide (AFBO) in vitro. Statistical analysis of the MTT results indicated that there were overall significant differences between the control and both the AFB1-treated (p < 0.0001) and AFBO-treated cells (p = 0.002). However, there was no significant difference between AFB1 and AFBO-treated cells, when the entire range of concentrations were assessed against each other (p = 0.2877). When analysed at each concentration, only at 0.01 mM was there a significant difference between the effects of AFB1 and AFBO (p = 0.0358). The results of this investigation show that AFB1 and AFBO are both cytotoxic in the A549 cell line.

Aqueous and methanol extracts of nine traditional Zulu medicinal plants, Cissus quandrangularis L., Cyphostemma flaviflorum (Sprague) Descoings, Cyphostemma lanigerum (Harv.) Descoings ex Wild & Drum, Cyphostemma natalitium (Szyszyl.) J. v. d. Merwe, Cyphostemma sp., Rhoicissus digitata (L. F.) Gilg & Brandt, Rhoicissus rhomboidea (E. Mey. Ex harv.) Planch, Rhoicissus tomentosa (Lam.) Wild & Drum, R. tridentata (L. F.) Wild & Drum and Rhoicissus tridentata (L. F.) Wild & Drum subsp. cuneifolia (Eckl. & Zeyh.) N. R. Urton, all belonging to the Vitaceae family, were evaluated to determine their therapeutic potentials as antineoplastic agents. The antiproliferative activity in vitro against HepG2 cells was determined. Twenty-two of the twenty-seven crude plant extracts showed activities ranging from 25% to 97% inhibition of proliferation when compared with the control which showed no inhibitory activity. Higher degrees of growth inhibition were found in aqueous root extracts in comparison with the methanol extracts of the same plant parts. The results show potential antineoplastic activity, indicating some scientific validation for traditional usage.

Fumonisin B1 (FB1) is a compound that occurs frequently in rural foods and feeds, creating health hazards. When ingested, FB1 does not appear to change in structure and is mostly excreted unchanged in faeces within 24 h. Twenty human stool samples obtained from rural school children of Vulamehlo, south of Durban (South Africa), were analysed for FB1, as well as 23 urban control samples obtained from various households within the Durban metropolitan area. The samples were freeze-dried and ground to a fine powder. A fraction of each sample was extracted three times with aqueous ethylenediaminetetraacetic acid at pH 5.2. The pooled extracts were purified using reversed phase C18 solid phase extraction cartridges. Analytical high performance liquid chromatography was used to quantitate the amount of FB1 as an o-phthaldialdehyde (OPA) derivative in the extracts. The rural (35%) and the urban samples (9%) showed the presence of FB ranging from 790 to 19 560 ng g(-1) of freeze dried stool. It was concluded that this method could be used as a routine biomarker for short term human exposure to FB1 in contaminated food.

Post exercise lymphocytopenia is well documented and attributed to egress of lymphocytes from the vascular compartment. Recent studies have reported exercise induced DNA damage in leukocytes and have questioned a possible link to apoptosis. Eleven subjects underwent a ramped treadmill test to exhaustion. Venous blood samples were taken before, immediately post exercise, and 24 and 48 hours after exercise. Single cell gel electrophoresis revealed evidence of single strand DNA damage in 10% of lymphocytes immediately after exercise, but not at other times. Fluorescent microscopy showed three patterns of DNA distribution, similar to those seen in apoptosis, at all times after exercise. Three subjects underwent the same exercise protocol, and lymphocytes were prepared for flow cytometry to determine apoptosis using the TUNEL method. Flow cytometry revealed lymphocyte apoptosis in 63% of lymphocytes immediately after exercise and 86.2%, 24 hours after exercise. Lymphocyte apoptosis is documented for the first time after exercise and may in part account for exercise induced lymphocytopenia and reduced immunity.

The effect of different doses of low intensity laser therapy (L.I.L.T.) on human fibroblasts was investigated to determine the optimal dose required to stimulate fibroblast proliferation. Human fibroblasts were cultured in vitro and irradiated with different energy densities of 83Onm continuous output infra-red laser using a Gallium Aluminium Arsenide laser. The fibroblasts were irradiated on three consecutive days at energy densities, ranging from 0.2 to 5 J.cm2, delivered at an average radiant power of 30 mW, and at a constant distance of lcm from the fibroblasts. Fibroblast activity was assessed on the fourth day using a calorimetric MTT (tetrazolium) cleavage assay. There was a significant increase in fibroblast proliferation at laser treatment energy densities of 0.4 J.cm2 and 5 J.cm2. Difficulties associated with in vivo and in vitro studies of the effect of laser treatment are discussed.

Seven alkyl and aryl homologues of O-methylsterigmatocystin (OMST) were synthesised and fed in separate experiments to a mutant of Aspergillus parasiticus capable of converting sterigmatocystin (ST) to aflatoxin B1 (AFB1). Their conversion to AFB1 was followed over a time period and it was found that O-propylsterigmatocystin (OPRST) was converted to AFB1 more rapidly than O-ethylsterigmatocystin (OEST) or OMST or ST itself. The aryl derivative O-benzoylsterigmatocystin (OBzST) was converted at the slowest rate. These results show that alkyl and aryl homologues of OMST may be converted to AFB1, suggesting that the methylation of ST is not an absolute requirement for its conversion to AFB1. It seems likely that whatever enzyme(s) are involved in this process exhibit relative specificity. As to whether alkylation of ST is an obligatory step in AFB1 biosynthesis is neither supported nor disproved as the fungal cells used are presumably capable of methylating ST. The fact that the propyl derivative showed fastest conversion is not necessarily significant as this may be due to faster diffusion of the least polar of the derivatives through the cell membrane.

Fumonisin B1 (FB1), a common mycotoxin contaminant of maize, is known to inhibit sphingolipid biosynthesis and has been implicated in cancer promoting activity in animals and humans. FB1 disrupts DNA methylation and chromatin modifications in human hepatoma (HepG2) cells. We investigated the effect of FB1 on enzymes, DNA methyltransferases and demethylases, involved in chromatin maintenance and gross changes in structural integrity of DNA in HepG2 cells. We measured: (i) the expression of 84 key genes encoding enzymes known to modify genomic DNA and histones (superarray and qPCR); (ii) protein expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and the major demethylase (MBD2) (western blotting); (iii) degree of DNA methylation by FACS using anti-5-MeCyt and (iv) DNA migration (single cell gel electrophoresis). FB1 significantly decreased the methyltransferase activities of DNMT1, DNMT3A and DNMT3B, and significantly up regulated the demethylases (MBD2 expression and activity, and KDM5B and KDM5C expression). FACS data showed FB1 significantly increased DNA hypomethylation and resulted in gross changes in structural DNA as evidenced by the Comet assay. We conclude that FB1 induces global DNA hypomethylation and histone demethylation that causes chromatin instability and may lead to liver tumourigenesis.

Background Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Promoter polymorphisms of these genes have been linked to chronic diseases including heart disease and type II diabetes mellitus in several populations. This is the first investigation of the UCP2 − 866G/A rs659366 and UCP3 − 55C/T rs1800849 polymorphisms in young South African (SA) Indians with coronary artery disease (CAD). Methods A total of 300 subjects were recruited into this study of which 100 were SA Indian males with CAD, 100 age- (range 24–45 years), gender- and race-matched controls and 100 age-matched black SA males. The frequency of the UCP2 − 866G/A and UPC3 − 55C/T genotypes was assessed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Results The heterozygous UCP2 − 866G/A and homozygous UCP3 − 55C/C genotypes occurred at highest frequency in CAD patients (60% and 64%, respectively) compared to SA Indian controls (52% and 63%) and SA Black controls (50% and 58%). The UCP2 − 886G/A (OR = 1.110; 95% CI = 0.7438–1.655; p = 0.6835) and UCP3 − 55C/T (OR = 0.788; 95% CI = 0.482–1.289; p = 0.382) polymorphisms did not influence the risk of CAD. The rare homozygous UCP3 − 55T/T genotype was associated with highest fasting glucose (11.87 ± 3.7 mmol/L vs. C/C:6.11 ± 0.27 mmol/L and C/T:6.48 ± 0.57 mmol/L, p = 0.0025), HbA1c (10.05 ± 2.57% vs. C/C:6.44 ± 0.21% and C/T:6.76 ± 0.35%, p = 0.0006) and triglycerides (6.47 ± 1.7 mmol/L vs. C/C:2.33 ± 0.17 mmol/L and C/T:2.06 ± 0.25 mmol/L, p Conclusion The frequency of the UCP2 − 866G/A and UCP3 − 55C/T polymorphisms was similar in our SA Indian and SA Black groups. The presence of the UCP2 − 866G/A and UCP3 − 55C/T polymorphisms does not influence the risk of CAD in young South African Indian CAD patients.

Mycotoxins that commonly contaminate staple food grains pose a health hazard to animals and humans. Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, causes equine leukoencephalomalacia and porcine pulmonary edema and has been implicated in the etiology of esophageal cancer (EC) in the Transkei, South Africa. Various studies have indicated that nitrosamines induce EC, and F. verticillioides enhancement of nitrosamine-induced EC in rats has been reported. Dietary catechol (CAT), a constituent of cigarette smoke, was previously found to be a cocarcinogen with methyl-N-nitrosamine for inducing esophageal tumors in rats. In the present study we therefore investigated the cytotoxic effects of FB1, diethylnitrosamine (DEN), and CAT on a human esophageal epithelial cell line (SNO) using the methylthiazol tetrazolium assay. For each treatment, toxin concentrations ranged from 2.165 to 34.64 micro M. The results showed that the cytotoxic response of SNO cells was highest in cells treated with 34.64 micro M FB1. SNO cells treated with DEN + FB1 showed greater cytotoxicity than did cells treated with FB1 alone, whereas FB1 appeared to inhibit the cytotoxic effect exerted by CAT alone. The results of this study provide further evidence for the involvement of FB1 in the etiology of esophageal carcinoma.

Background. Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in coronary artery disease (CAD).Objectives. We aimed to assess 2 common polymorphic variant isoforms in GSTM1 and GSTP1 of GST in young CAD patients.Methods. All patients (N=102) were South Africans of Indian ancestry, a population associated with high CAD risk. A corresponding age-, sex- and race-matched control group (N=100) was also recruited. Frequency of the GSTM1 +/0 (v. +/0 and 0/0) and GSTP1 A105/G105 (v. wild-type A105/A105) genotypes was assessed by differential polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP), respectively.Results. The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% v. 18% and 65% v. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (odds ratio (OR)=2.593; 95% confidence interval (CI) 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 OR=0.6011; 95% CI 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 relative risk (RR)=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221).Conclusion. Our findings support the association of genotypes GSTM1 0/0 and GSTP1 A105/A105 and smoking with CAD.S Afr Med J 2012;102(7):627-630.

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease, which causes synovial damage. Persistence of lymphocyte infiltrates in the rheumatoid synovium has been attributed to abnormal apoptosis. While not comprehensively investigated, perturbations in peripheral blood lymphocyte (PBL) apoptosis may also be involved in perpetuation of autoimmune processes in RA. Methods We investigated total, CD4+ and CD19+ PBL apoptosis in our study cohort by monitoring the translocation of phosphatidylserine using the Annexin-V assay. To examine the role of death receptor mediated apoptosis as well as activation-induced-cell-death (AICD), PBLs were labeled with CD95/Fas and CD69 markers and enumerated by flow cytometry. Proteolytic activity of initiator and executioner caspases was determined by luminometry. DNA fragmentation assays were used to examine whether apoptotic signals were transduced to the nucleus. Quantitative PCR arrays were used to investigate apoptotic pathways associated with RA-PBLs. Since heat-shock-protein-70 (HSP70) is an inducible protein which modulates apoptotic signals, we determined HSP70 levels by intra-cellular flow cytometry and western blots. Results The RA-PBLs showed signs of elevated apoptosis whilst in circulation. These include increases in the loss of plasma membrane asymmetry, indicated by increased externalization of phosphatidylserine (especially in B-lymphocytes). RA-PBLs showed a bias to CD95/Fas mediated apoptotic pathways, but low levels of the CD69 marker suggested that this was not associated with immune activation. Although downstream markers of apoptosis such as caspase-3/7 activity, were increased, no DNA fragmentation was observed in RA-PBLs. Interestingly, elevated levels of apoptosis did not correlate with absolute lymphocyte counts in RA patients. Levels of HSP70 were highly elevated in RA-PBLs compared to controls. Conclusion The results suggest that while apoptosis may be initiated in RA-PBLs, they may lack commitment to fully executing the apoptotic program. This may be related to inhibition on apoptotic transduction by HSP70. This study provides evidence that abnormalities in RA-PBLs apoptosis may occur whilst still in circulation and may contribute to pathogenesis of the disease.

Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa, is traditionally used to treat a diverse range of illnesses. More specifically, the immune-enhancing potential of SF has been recognised to the extent that SF extracts have been recommended as an adjuvant in HIV/AIDS treatment by the South African Ministry of Health, despite a lack of knowledge of its mechanism of action or potential immune toxicity. As yet, unsubstantiated data support the notion of immunostimulatory effects of SF extracts in HIV-infected patients. This was suggested by post-treatment recovery of CD4+ cells brought about by the reduction of the impact of virus-induced apoptosis. This study investigated the apoptotic effects of SF extracts on normal human lymphocytes in vitro. Initially, an acute cytotoxic profile of SF extract was formulated, from which an IC50 of 7.5 mg/mL was calculated and administered for 3 h, 6 h and 12 h to cell populations. At 12 h, SF caused a significant increase in apoptosis in the total lymphocyte population and CD4+ cells as evidenced by increased phosphatidylserine (PS) translocation, caspase-3/7 activity, and decreased ATP content. After 12 h, the SF extract initiated lymphocyte activation in both total lymphocyte and CD4+ subpopulations, indicated by a doubling of the number of cells expressing the CD69 activation marker. The apoptosis observed may thus be the result of activation-induced lymphocyte cell death (AICD). Our results are in conflict with preliminary clinical evidence which has suggested SF extracts are possibly beneficial in the treatment of HIV infection. More extensive evaluations of the effects of SF extracts on the immune system in such subjects are urgently needed.

The p53 tumor-suppressor protein plays an integral role in apoptosis. Perturbations in peripheral lymphocyte (PL) apoptosis may be associated with rheumatoid arthritis (RA). Polymorphisms at codon 72 of p53 (arginine (Arg72) to proline transition) confers differences in mitochondrial translocation and apoptosis inducing capabilities of p53 in vitro. We examined associations of this polymorphism with PL apoptosis, mitochondrial depolarization, and clinical markers of disease activity in a cohort of black South African RA patients. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. PL apoptosis was measured using the annexin-V assay and mitochondrial membrane potential with the JC-1 assay. Clinical and laboratory parameters were recorded for all patients. Statistical differences in these parameters were investigated according to genotype. Genotype distribution did not differ significantly between RA patients and controls (Arg/Arg, Arg/Pro, Pro/Pro: 12%, 46%, and 42% versus 3%, 34%, and 63%), despite significantly higher frequency of the Arg72 allele in patients (p = 0.0406). There was no significant difference in PL apoptosis and mitochondrial depolarization based on p53 codon 72 genotype. In addition, clinical markers of disease activity were not significantly different between genotypes. We conclude that p53 codon 72 genotype does not influence PL apoptosis or mitochondrial depolarization and is not associated with clinical markers of disease in RA.

The activity of the enzyme responsible for the conversion of norsolorinic acid to averantin was studied in two strains of Aspergillus parasiticus. Cell-free extracts of the enzyme were purified from different aged mycelia and little activity was found prior to 24 hours after inoculation but this quickly reached a maximum at 48 hours and declined thereafter. Both strains of A. parasiticus, one in aflatoxin producing strain, the other a versicolorin A accumulating mutant, showed this trend. It was concluded that the enzyme responsible for this conversion was a secondary metabolic enzyme and was distinct from alcohol and mannitol dehydrogenases.

On administration of aflatoxin B1 to whole parsley (Petroselinum crispum) plants, a derivative was formed, which was shown to be aflatoxicol by its chromatographic properties and mass spectrometry. Optimum conditions for the production of the derivative was on the second day after administration of the toxin to the plants, which were 90 days old after germination. Cell-free preparations of parsley were found not to produce aflatoxicol A from added aflatoxin B1; instead they formed two new derivatives, which from chromatographic properties, were shown to be more polar than either aflatoxin B1 or aflatoxicol A.

An affinity matrix for the purification of norsolorinic acid dehydrogenase, an enzyme involved in aflatoxin biosynthesis, was prepared by coupling norsolorinic acid to an agarose gel. This matrix was found to be ineffective in isolating active enzyme, and was therefore modified by methylation, using diazomethane. The methylated matrix produced a one-step purification of the enzyme from a crude homogenate, resulting in a 138-fold purification. The active isolate was found to contain one major and two minor bands upon nondenaturing electrophoresis, and all the norsolorinic acid dehydrogenase activity was associated with the major band. It was concluded that the matrix exhibited true affinity for the enzyme, and that affinity chromatography was a valuable approach to isolating other secondary metabolic enzymes involved in the biosynthesis of the aflatoxins.

Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O-methylsterigmatocystin (OMST) to the potent environmental carcinogen aflatoxin B1 (AFB1), has been proposed to be catalysed by an oxidoreductase (OR) that requires a cytochrome P-450 type of oxidoreductase activity. This enzyme displays relative specificity towards OMST homologues in fungal whole cells. These studies were extended to the action of a cell-free enzyme system (CFES), on five OMST homologues, with a view to establish the kinetics. In the current study a CFES, containing an oxidoreductase, was derived from a blocked mutant of Aspergillus parasiticus (Wh1-11-105). The key experimental steps involved rapid concentration and efficient dialysis by membrane filtration to remove small biomolecules (MW10,000), co-factors, primary and secondary metabolites. The kinetic parameters of the enzyme-substrate reactions indicated that the reaction follows a Michealis-Menten kinetics and OR activity decreased in the order: O-butylsterigmatocystinO-propylsterigmatocystinO-ethylsterigmatocystinO-methylsterigmatocystinO-acetylsterigmatocystinO-benzoylsterigmatocystin. The 7-O-alkyl homologues were the best substrate for the CFES, thereby substantially supporting that the 7-O-methyl group of OMST is preferred for OR catalytic activity in the absence of any other alkylating groups in vitro. The Km was calculated as 5.65 microM for this CFES and varied marginally among the OMST homologues studied.

Zearalenone (6-[10-hydroxy-6-oxo-trans-1-undecenyl]-beta-resorcylic acid-p-lactone), a mycotoxin produced in corn, is able to adopt a conformation which sufficiently resembles 17beta-oestradiol to allow it to bind to the oestrogen receptor in target cells of the body and exert (agonist) oestrogenic action. We adopted an analytical approach to isolate and quantify the mycotoxin and its derivatives using high-performance liquid chromatography and gas chromatography-mass spectrometry. In this study, the quantity of zearalenone and its congeners (alpha-zearalenol and beta-zearalenol) present in the plasma of patients with breast (n = 28) and cervical carcinoma (n=54) were compared with levels in patients presenting with other diagnoses (n = 26) and healthy volunteers (n = 24). There were no significant differences between the groups in the levels of zearalenone and its congeners, using analysis of covariance (0.2p0.6). These results suggest that the presence of this mycotoxin in blood does not indicate causal relationship between exposure to this exogenous myco-oestrogen and the subsequent biological effect in our study population but may be used as an indicator of exposure. The presence of zearalenone in the study groups is, however, of growing concern, due to the possible effects of cumulative long-term exposure of oestrogenic target organs to this compound.

Background The incidence of lung cancer is expected to increase due to increases in exposure to airborne pollutants and cigarette smoke. Moringa oleifera (MO), a medicinal plant found mainly in Asia and South Africa is used in the traditional treatment of various ailments including cancer. This study investigated the antiproliferative effect of MO leaf extract (MOE) in cancerous A549 lung cells. Methods A crude aqueous leaf extract was prepared and the cells were treated with 166.7 μg/ml MOE (IC50) for 24 h and assayed for oxidative stress (TBARS and Glutathione assays), DNA fragmentation (comet assay) and caspase (3/7 and 9) activity. In addition, the expression of Nrf2, p53, Smac/DIABLO and PARP-1 was determined by Western blotting. The mRNA expression of Nrf2 and p53 was assessed using qPCR. Results A significant increase in reactive oxygen species with a concomitant decrease in intracellular glutathione levels (p p p p p p Conclusion MOE exerts antiproliferative effects in A549 lung cells by increasing oxidative stress, DNA fragmentation and inducing apoptosis.

Background Silver nanoparticles (AgNP), the most popular nano-compounds, possess unique properties. Albizia adianthifolia (AA) is a plant of the Fabaceae family that is rich in saponins. The biological properties of a novel AgNP, synthesized from an aqueous leaf extract of AA (AAAgNP), were investigated on A549 lung cells. Cell viability was determined by the MTT assay. Cellular oxidative status (lipid peroxidation and glutathione (GSH) levels), ATP concentration, caspase-3/-7, -8 and −9 activities were determined. Apoptosis, mitochondrial (mt) membrane depolarization (flow cytometry) and DNA fragmentation (comet assay) were assessed. The expression of CD95 receptors, p53, bax, PARP-1 and smac/DIABLO was evaluated by flow cytometry and/or western blotting. Results Silver nanoparticles of AA caused a dose-dependent decrease in cell viability with a significant increase in lipid peroxidation (5-fold vs. control; p = 0.0098) and decreased intracellular GSH (p = 0.1184). A significant 2.5-fold decrease in cellular ATP was observed upon AAAgNP exposure (p = 0.0040) with a highly significant elevation in mt depolarization (3.3-fold vs. control; p AgNP treated cells (p p = 0.0416). Silver nanoparticles of AA caused a significant 2.5-fold reduction in caspase-8 activity (p = 0.0024) with contrasting increases in caspase-3/-7 (1.7-fold vs. control; p = 0.0180) and −9 activity (1.4-fold vs. control; p = 0.0117). Western blots showed increased expression of smac/DIABLO (4.1-fold) in treated cells (p = 0.0033). Furthermore, AAAgNP significantly increased the expression of p53, bax and PARP-1 (1.2-fold; p = 0.0498, 1.6-fold; p = 0.0083 and 1.1-fold; p = 0.0359 respectively). Conclusion Data suggests that AAAgNP induces cell death in the A549 lung cells via the mt mediated intrinsic apoptotic program. Further investigation is required to potentiate the use of this novel compound in cancer therapy trials.