Adverse effects of copper, manganese and mercury, alone and in mixtures on the aorta and heart of Spraque-Dawley rats.

Cardiovascular diseases (CVD) are a common global cause of death and are therefore a major health concern. Inhaled or ingested environmental heavy metals contribute to the development of CVD. The aim of this study was to address the limited information available on the effect of relevant dosages of metals in mixtures. Three metals with reported effects on the cardiovascular system (CVS) were identified, and these metals were copper (Cu), manganese (Mn) and mercury (Hg). In Sprague-Dawley rats, the adverse effects of copper (Cu), manganese (Mn) and mercury (Hg), alone and as part of mixtures, on the blood parameters, the aorta and heart were investigated. Forty-eight male Sprague-Dawley rats were randomly divided into eight groups (n = 6): control, Cu, Mn, Hg, Cu + Mn, Cu + Hg, Mn + Hg and Cu, Mn + Hg. The seven experimental groups received the metal mixtures at 100 times the World Health Organisation (WHO) safety limit for drinking water (2 mg/L for Cu, 0.4 mg/L for Mn and 0.06 mg/L for Hg) via oral gavage for 28 days. After 28 days, compared with the control, red blood cell levels were increased for Cu + Hg. All other measured blood parameters were unchanged. Morphological changes in the tunica media were connective tissue deposition and an abundance of collagen type I in the metal exposed aortic tissues. In the cardiac tissue of metal-exposed rats, changes in the cardiomyocyte and myofibrillar arrangement, with an increase in collagen type I and III was observed. Ultrastructurally, the aortic collagen and elastin band arrangement and the cardiac mitochondrial and myofibrillar arrangement and structures were altered in the experimental groups. These changes indicated that exposure to these metals in rats caused minor changes in the blood parameters, however, the changes in tissue and cellular structure indicated an increased risk for the development of CVD.

The potential antidiabetic properties of green and purple tea [Camellia sinensis (L.) O Kuntze], purple tea ellagitannins, and urolithins.

ETHNOPHARMACOLOGICAL RELEVANCE: Tea (Camellia sinensis) has been consumed for centuries as traditional medicine for various diseases, including diabetes. The mechanism of action of many traditional medicines, including tea, often requires elucidation. Purple tea is a natural mutant of Camellia sinensis, grown in China and Kenya, and is rich in anthocyanins and ellagitannins. AIM OF THE STUDY: Here we aimed to determine whether commercial green and purple teas are a source of ellagitannins and whether green and purple teas, purple tea ellagitannins and their metabolites urolithins have antidiabetic activity. MATERIALS AND METHODS: Targeted UPLC-MS/MS was employed to quantify the ellagitannins corilagin, strictinin and tellimagrandin I, in commercial teas. The inhibitory effect of commercial green and purple teas and purple tea ellagitannins was evaluated on α-glucosidase and α-amylase. The bioavailable urolithins were then investigated for additional antidiabetic effects, by evaluating their effect on cellular glucose uptake and lipid accumulation. RESULTS: Corilagin, strictinin and tellimagrandin I (ellagitannins) were identified as potent inhibitors of α-amylase and α-glucosidase, with Ki values significantly lower (p < 0.05) than acarbose. Commercial green-purple teas were identified as ellagitannin sources, with especially high concentrations of corilagin. These commercial purple teas, containing ellagitannins, were identified as potent α-glucosidase inhibitors with IC50 values significantly lower (p < 0.05) than green teas and acarbose. Urolithin A and urolithin B were as effective (p> 0.05) as metformin in increasing glucose uptake in adipocytes, muscle cells and hepatocytes. In addition, similar (p > 0.05) to metformin, both urolithin A and urolithin B reduced lipid accumulation in adipocytes and hepatocytes. CONCLUSIONS: This study identified green-purple teas as an affordable widely available natural source with antidiabetic properties. Furthermore, additional antidiabetic effects of purple tea ellagitannins (corilagin, strictinin and tellimagrandin I) and urolithins were identified.

The impact of boiling and in vitro human digestion of Solanum nigrum complex (Black nightshade) on phenolic compounds bioactivity and bioaccessibility.

Solanum nigrum complex (Black nightshade) is a wild leafy vegetable with phenolic antioxidant compounds related to the reduction of oxidative stress. Changes in phenolics and bioactivity due to cooking and gastrointestinal digestion of black nightshade were compared to spinach. Predominant compounds of black nightshade were myricetin, quercetin-3-O-robinoside, 3,4-dicaffeoylquinic acid, 3-caffeoylquinic acid, and rutin, which were improved after boiling but reduced after in vitro digestion. Phenolics were reduced after digestion of black nightshade and spinach; however, bioactivity was still retained, especially in preventing oxidative stress in Caco-2 cells. Hence, indicating their potential to reduce oxidative stress related diseases of the digestive tract.

Influence of boiling and subsequent phases of digestion on the phenolic content, bioaccessibility, and bioactivity of Bidens pilosa (Blackjack) leafy vegetable.

The effects of boiling and in vitro human simulated digestion on phenolic compounds and bioactivity of the African leafy green vegetable, Bidens pilosa, known as Blackjack in South Africa, was investigated and compared to the leafy green vegetable Spinacia oleracea (Spinach). Metabolites such as 3-caffeoylquinic acid, catechin, 4-caffeoylquinic acid, quercetin-3-O-robinobioside, rutin and quercetin-3-O-glucoside were dominant in the boiled methanolic compared with the raw methanolic extracts of the Blackjack. The total phenolic and flavonoid content generally decreased after in vitro complete digestion for both raw and boiled extracts, indicating that pH and environment associated with digestion alters the bioactivity of the extracted phenolics. Both leafy green vegetables had beneficial effects, but all Blackjack extracts were more effective in preventing the AAPH-mediated oxidation of Caco-2 cells, low-density lipoprotein and deoxyribonucleic acid than those of the spinach. This study identified the health benefits of eating Blackjack and therefore, the cultivation and consumption of this leafy green vegetable should be promoted.

Oxidative and haemostatic effects of copper, manganese and mercury, alone and in combination at physiologically relevant levels: An ex vivo study.

Water contamination with metals due to anthropogenic activity is increasing and subsequent exposure increases the risk of associated toxicity. Exposure is not limited to a single metal but usually involves mixtures of different metals at different concentrations. Little is known about the contribution of this type of exposure, in humans, to the development of non-communicable diseases such as cardiovascular disease, and an increased risk to thrombosis. The World Health Organization has established limits for metal levels in drinking water and this includes levels for copper (Cu), manganese (Mn) and mercury (Hg). In this study, at 100X these limits, the ability of the metals' oxidative effects as catalysts of the Fenton reaction and/or ability to bind glutathione (GSH) were determined. The haemostatic effects of these metals, alone and in combination, at the World Health Organization limit were then evaluated. The ultrastructural and viscoelastic alterations of exposed ex vivo whole blood were also evaluated using scanning electron microscopy and thromboelastography® (TEG), respectively. Cu, alone and in combination with Mn and/or Hg, induced hydroxyl radical formation and reduced GSH levels. Ex vivo exposure caused deformation of erythrocytes and accelerated platelet activation especially for Cu, alone and in combination, with Mn. Reduction in the lysis potential of the clot was also observed for all combinations, especially Cu in combination with Hg as well as Mn alone. Although the TEG findings were not statistically significant, the trends indicate that the exposure to these metals, alone and in combination, adversely affects thrombus formation in ex vivo blood, thereby potentially increasing the risk in exposed individuals for thrombosis.

Exploring the anti-proliferative activity of Pelargonium sidoides DC with in silico target identification and network pharmacology.

Pelargonium sidoides DC (Geraniaceae) is a medicinal plant indigenous to Southern Africa that has been widely evaluated for its use in the treatment of upper respiratory tract infections. In recent studies, the anti-proliferative potential of P. sidoides was shown, and several phenolic compounds were identified as the bioactive compounds. Little, however, is known regarding their anti-proliferative protein targets. In this study, the anti-proliferative mechanisms of P. sidoides through in silico target identification and network pharmacology methodologies were evaluated. The protein targets of the 12 phenolic compounds were identified using the target identification server PharmMapper and the server for predicting Drug Repositioning and Adverse Reactions via the Chemical-Protein Interactome (DRAR-CPI). Protein-protein and protein-pathway interaction networks were subsequently constructed with Cytoscape 3.4.0 to evaluate potential mechanisms of action. A total of 142 potential human target proteins were identified with the in silico target identification servers, and 90 of these were found to be related to cancer. The protein interaction network was constructed from 86 proteins involved in 209 interactions with each other, and two protein clusters were observed. A pathway enrichment analysis identified over 80 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched with the protein targets and included several pathways specifically related to cancer as well as various signaling pathways that have been found to be dysregulated in cancer. These results indicate that the anti-proliferative activity of P. sidoides may be multifactorial and arises from the collective regulation of several interconnected cell signaling pathways.

Effects of Mandrax and Cannabis on the cellular function of chick embryonic neurons.

Cannabis and Mandrax abuse is unique to South Africa; and most research has focused on the socio-economic impact rather than the adverse effects on the developing brain. Therefore, the aim of this study is to determine the effects of Mandrax and Cannabis alone and in combination on the developing brain by using primary and suspension cultures of the chick embryo brain. Exposure of primary chick embryo neuronal (CEN) cultures to the carrier ethanol, Mandrax and Cannabis, for 24h resulted in a significant dose dependent decrease in cell number for Mandarx alone. Increasing concentrations of Cannabis in combination with Mandrax inhibited the toxic effect of Mandrax. In CEN suspensions, Mandrax alone induced a significant time-concentration dependent decrease in esterase activity following 1 and 4h exposure. In combination with Cannabis, a significant increase in esterase activity was observed after 4h exposure. In conclusion Mandrax is toxic to CEN cells in vitro while Cannabis seemed to have a protective effect; however, this study does not investigate the abuse of these drugs in the form commonly abused, namely inhaled smoke.

Effects of Urginea sanguinea, a traditional asthma remedy, on embryo neuronal development.

The Southern African plant, Urginea sanguinea Shinz (Hyacinthaceae) (US), is a well-known traditional herbal medicine and it is used for many different ailments, including asthma. Pregnant women also use this plant and little is known regarding the toxic effects of this plant material on the developing foetus. US contains the cardiac glycoside (CG) Transvaalin; CGs are known to cross the placenta and blood-brain barrier and therefore may have a negative effect on the foetal development. To address this, in vitro cytotoxicity of this preparation as well as its effect on chick embryo neural development was investigated. Water extracts of US were shown to be cytotoxic in cell cultures of L929 cell and primary embryonic neural cell cultures. Electron microscopy studies following in ovo exposure revealed altered neuron morphology with patterns of cell damage either associated with apoptosis or necrosis. CGs are known to inhibit membrane bound Na(+)/K(+)-ATPase in conducting tissues, causing disruption of the calcium pathways, mitochondrial calcium overload leading to either apoptosis or necrosis or where both occur, a process of necrapoptosis. The in ovo effects observed strongly indicate that US causes necrapoptosis in chick embryonic neurons.

Urginea sanguinea: medicinal wonder or death in disguise?

The Southern African plant, Urginea sanguinea Shinz (Hyacinthaceae) is well-known for its poisonous affects on livestock, but it is widely used as herbal medicines by traditional healers. It is most frequently used as a blood purifier, abortifacient, treatment for venereal diseases, abdominal pain, backache and hypertension. In this article we discuss the plant's morphology, distribution, medicinal values but also poisonous characteristics. Furthermore, we discuss the most important chemical components, with special reference to the significance and physiological action of the cardiac glycoside (CG), Transvaalin. Emphasis is placed on the physiological mechanism of CG toxicity involving the Na(+)/K(+)-ATPase pump. This pump is of extreme importance, especially in conducting tissues such as nerve fibers, cardiac-, skeletal- and smooth muscles. Furthermore, we summarize the medicinal uses, the systems it influence as well the adverse effects in case of over dosage.

Presenting an in vitro cell culture model to determine the cytotoxic effect of benzoyl peroxide vapours.

Benzoyl peroxide (BP) is an initiator of polymerisation used in the synthesis of methyl methacrylate dental materials, and is a known allergen that causes allergic contact dermatitis during occupational exposure, especially in dentists and dental personnel. The eyes of dental technicians, dentists and even patients with an allergic history are the first level of exposure, and therefore complaints of allergic reactions of the eyes are usually noted. In this article the authors used a modification direct cell culture testing method to assess the cytotoxic potential of BP. L929 mouse fibroblasts as well as cells from human limbal eye rings were exposed to BP vapours in the experiments. The MTT and crystal violet assays were also employed to determine cell numbers and viability. Results indicated that there is an exponential decrease in cell viability. After exposure of five minutes, cell viability had already decreased by 20 and 40% for vapours derived from either a 10 or 20 microliters aliquot of BP, respectively. After a further five minutes, cell viability decreased by a further 10% with a statistical difference form the control of p < 0.01. Results indicated that BP vapours are cytotoxic to mouse L929 and human eye fibroblasts. Furthermore, the authors concluded that the permanent L929 mouse fibroblast cell line can provide valuable information regarding the cytotoxic effects of dental products and associated compounds that form toxic vapours.

An in vitro study of biological safety of condoms and their additives.

The use of condoms to prevent sexually transmitted diseases, especially HIV, is widely encouraged. Condoms contain latex, nonspermicidal lubricants (such as dimethylsiliconium) and other nonspecified compounds, such as colorants and flavorings. Latex causes allergy reaction in susceptible individuals but little is known regarding the cytotoxic effects of other additives. The objective of this study was to develop a sensitive in vitro system to determine the toxic effects of condom material. The modified L929 FDA method and a more specific cell type, such as the cervical epithelial tumor cell line HeLa, was used. Lubricated (LC), lubricated and flavored (LFC), and lubricated, flavored and colored condoms (LFCC) were evaluated. Washings containing condom surface material were prepared by washing condom fragments in medium for different time intervals. Changes in cell number, viability and lysosome integrity in the L929 and HeLa cell lines was determined using the Crystal Violet, MTT and Neutral Red assays, respectively. The condom type affected cell viability and lysosome integrity, with LC inducing an increase in cell viability and LFC a decrease in lysosome integrity. The HeLa cell line in combination with the MTT and NR assay was themost sensitive in vitro system to determine the toxic effects of condom material.

Novel test and its automation for the determination of erythrocyte acetylcholinesterase and its application to organophosphate exposure.

BACKGROUND: Because of the lack of a problem-free, reliable method for determination of erythrocyte acetylcholinesterase (AChE), we developed a simple kinetic method, which we found to be both reliable and suitable for automation in the routine clinical laboratory. METHODS: Acetylthiocholine, used as substrate, is hydrolysed by acetylcholinesterase to yield acetate and thiocholine. Thiocholine reacts with dichlorophenolindophenol, a blue coloured compound, which is reduced to a colourless product, producing a linear decrease in absorption at 606 nm. If required, this assay can also be run at 600 nm with equally acceptable results. RESULTS: The method was automated on the Synchron LX20 multianalyser (Beckman Instruments) and blood samples of 80 patients with clinically symptomatic organophosphate poisoning and 153 normal controls were evaluated. Acetylcholinesterase values were in the range of 0-14 UgHb(-1) in cases of organophosphate poisoning, in contrast with normal controls, who had AChE values of 24.4--37.9 UgHb(-1). No overlap was found between AChE values of controls and poisoned cases. Intra- and inter-assay coefficients of variation were 1.68 and 3.71%, respectively. CONCLUSION: The method we propose for measurement of AChE was found to be simple, reliable and easily automatable in the routine clinical laboratory.

The biological safety of condom material can be determined using an in vitro cell culture system.

Latex products have long been recognized as a cause of latex protein allergy. The increased usage of latex gloves, with the consequent increased occurrence of latex allergies appears to have escalated with increasing awareness of the transmission of HIV-AIDS and other infections. The use of condoms as a means to prevent the transmission of STD's (sexually transmitted diseases) and HIV-AIDS has been widely promoted. Although extensive testing is done to evaluate the physical quality of condoms, no information is available regarding the biological safety of condoms. This study was undertaken to determine the effects of short-term exposure to physiological levels of condom surface material on cell viability (MTT assay) and cell growth (crystal violet assay). A direct contact cell culture testing method (FDA test method F813-83 used to evaluate the cytotoxic potential of medical materials and devices) was used. The modified test method was found to be a sensitive test system for the evaluation of the biological safety of condoms. This study reveals the importance of evaluating the biological safety of all condoms that are commercially available, because of the potential health risk that may be associated with prolonged use of certain types of condoms.

Folate status, homocysteine metabolism, and methylene tetrahydrofolate reductase genotype in rural South African blacks with a history of pregnancy complicated by neural tube defects.

The birth incidence of neural tube defects (NTDs) in South Africa is threefold to sixfold higher in rural compared with urban blacks. We investigated whether folate deficiency and aberrant homocysteine metabolism could explain the high NTD incidence in rural black populations. Plasma folate and total homocyst(e)ine (tHcy) concentrations were determined in apparently healthy rural black women (n = 107), rural black women with a history of pregnancy complicated by NTDs (n = 54), and urban blacks (n = 101). Methionine load tests were performed on the 54 women with a history of NTD-affected pregnancy and 54 controls matched for age and body mass. The presence of the 677C --> T mutation in the methylene tetrahydrofolate reductase (MTHFR) gene was investigated in both groups by a polymerase chain reaction (PCR) of genomic DNA and HinfI digestion of the PCR product. Apparently healthy urban black women (n = 101) had a lower (P < .001) plasma folate concentration compared with rural black women (n = 107). Women with a history of NTD-affected pregnancy did not differ significantly from controls with respect to plasma folate, fasting homocyst(e)ine, methionine, and the post-methionine load increase in plasma homocyst(e)ine. More than 50% of both of the latter groups had a post-methionine load increase in plasma tHcy less than the fifth percentile as observed in a healthy white control group. No homozygotes for the 677C --> T mutation in the MTHFR gene were found in black mothers with NTD-affected offspring or controls. It is concluded that black urbanization is characterized by a diminished folate status that is paradoxically associated with a lower NTD birth incidence. Homozygosity for the 677C --> T mutation in the gene coding for MTHFR does not constitute a genetic risk factor for NTDs in blacks. No aberrant homocysteine metabolism could be demonstrated in black women with NTD-affected pregnancies.

Spontaneous oxidation of methionine: effect on the quantification of plasma methionine levels.

Plasma methionine (Met), methionine sulfoxide (MSO), and total Met concentrations were determined by reversed-phase chromatography and fluorescence detection after automated precolumn derivatization with an o-phthalic aldehyde mercaptoethanol reagent. Addition of pure, MSO-free L-Met to plasma samples resulted in the anticipated linear increase in plasma Met concentrations, but simultaneously effected a dose-dependent, linear increase in MSO levels. In contrast, the addition of pure L-MSO to plasma samples rendered linear calibration curves for MSO, while the Met concentration remained constant. A strong buffering effect against the spontaneous or hydrogen peroxide induced oxidation of Met to MSO was observed in plasma samples. This protective effect could be neutralized by preincubating the plasma samples with sodium azide. The addition of relatively low concentrations of red cell lysates to plasma samples, prior to hydrogen peroxide oxidation, strongly inhibited the conversion of Met to MSO. Plasma samples from 127 healthy female volunteers were analyzed: MSO concentrations (mean, 3.6 +/- 2.1 microM) exhibited a weak positive correlation (r = 0.352) with Met levels (mean, 21.3 +/- 6.1 microM) but, after the exclusion of two probable outliers from the data set, no correlation was observed. Our results suggest that plasma Met concentrations should be corrected for oxidative losses incurred during storage, sample processing and because of the action of a variety of in situ oxidants, present in plasma, in order to obtain a reliable estimate of the methionine status of an individual.

Oestrogen and progesterone receptor status and PgR/ER ratios in normal and myomatous human myometrium.

The concentration and molecular properties of the oestrogen (ER) and the progesterone (PgR), present in normal myometria and uterine leiomyometria, obtained from a group of age-matched, pre-menopausal, negroid female patients were investigated. Serum oestrogen and progesterone levels did not differ significantly in the two groups. Significant differences were detected in ER and PgR levels between normal and leiomyomatous myometria. Both ER (154%; p < 0.0001) and PgR (33%; p < 0.05) were significantly increased in uterine leiomyomas. PgR levels were less affected than the ER levels, causing a significant decrease (44%; p < 0.05) in the PgR/ER ratio in myomatous myometria. Dissociation and sedimentation constants, as well as iso-electric points of ER and PgR were essentially similar in normal and in myomatous myometria. According to our results, tissue pathology does not appear to be associated with defects in the molecular properties of ER and PgR, but with differential changes in the ER and PgR levels, subsequently affecting the PgR/ER ratio.

Imaging infection and inflammation in an African environment: comparison of 99Tcm-HMPAO-labelled leukocytes and 67Ga-citrate.

A considerable segment of the population of South Africa has a disease spectrum similar to those of the industrialized countries. However, a much larger proportion of its inhabitants comes from historically disadvantaged communities and is plagued by diseases typical of the developing world. Infectious diseases head the list as a cause of mortality in certain age groups. We have studied 108 patients presenting with a wide array of infections. Fifty-eight patients were examined with 67Ga-citrate and with 99Tcm-HMPAO-labelled leukocytes, 40 with leukocytes alone and 10 who had a high pre-test probability of tuberculosis with 67Ga only. The sensitivity and specificity of 99Tcm-labelled leukocyte scintigraphy in the 58 patients who had both studies were 89 and 84%, respectively, while the corresponding values for 67Ga scintigraphy were 81 and 74%, respectively. The overall sensitivity and specificity for 99Tcm-labelled leukocytes for detecting inflammatory sites in all 98 cases were 92 and 89%, respectively. Although labelled leukocytes were the better of the two agents for the early diagnosis of infections with a high probability of neutrophil infiltration, 67Ga remains an excellent alternative. It is the first choice in patients in whom tuberculosis or a neoplastic process is suspected as a cause of fever. It is also safer to use in areas where labelling of leukocytes is inadvisable because of inadequate sterility or where the risk is high of infecting other patients or staff with HIV.

Cholate and pH reduce interference by sodium dodecyl sulfate in the determination of DNA with Hoechst.

The use of the fluorescent dye 33258 Hoechst (Hoe) to quantitatively determine DNA in cell culture in the presence of lysing agents like sodium dodecyl sulfate (SDS) is limited by the masking effect of high levels of nonspecific fluorescence, caused by the binding of Hoe to micelles. The masking effect can be reduced substantially by increasing the concentration of the counterion, the addition of cholate, or the pH of the buffer. An optimized method was developed, combining the antimasking effects of sodium chloride, cholate, and pH to accurately determine DNA concentrations as low as 15 ng/ml in the presence of up to 6.9 mM (0.2%) SDS. The effectiveness of SDS in cell dissolution can now be combined with the specificity and sensitivity of Hoe to determine cellular DNA.