Underlying mechanisms of cytotoxicity in HepG2 hepatocarcinoma cells exposed to arsenic, cadmium and mercury individually and in combination.

BACKGROUND: Toxicity data regarding combinational exposure of humans to arsenic, cadmium and mercury is scarce. Although hepatotoxicity has been reported, limited information is available on their mechanistic underpinnings. The cytotoxic mechanisms of these metals were determined in HepG2 hepatocarcinoma cell lines after individual and combinational exposure. METHODS: HepG2 cells were exposed to heavy metals (sodium arsenite, cadmium chloride, and mercury chloride) individually or in combination for 24 h, after which cell density, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), reduced glutathione (GSH), adenosine triphosphate (ATP) and caspase-3/7 activity was assessed. RESULTS AND DISCUSSION: Cadmium (IC50 = 0.43 mg/L) and the combination (0.45 mg/L, arsenic reference) were most cytotoxic, followed by arsenic (6.71 mg/L) and mercury (28.23 mg/L). Depolarisation of the ΔΨm and reductions in ROS, GSH and ATP levels occurred. Arsenic, cadmium and the combination increased caspase-3/7 activity, while mercury reduced it. CONCLUSION: The combination produced a greater, albeit mechanistically similar, cytotoxicity compared to individual metals. Cytotoxicity was dependent on altered mitochondrial integrity, redox-status, and bioenergetics. Although the combination's cytotoxicity was associated with caspase-3/7 activity, this was not true for mercury. Heavy metal interactions should be assessed to elucidate molecular underpinnings of cytotoxicity.

Use of Yokukansan (TJ-54) in the treatment of neurological disorders: a review.

Kampo herbal remedies are reported to have a wide range of indications and have attracted attention due to reports suggesting that these remedies are effective when used in disease treatment while maintaining a favourable quality of life. Yokukansan, also known as TJ-54, is composed of seven herbs; Angelica acutiloba, Atractylodes lancea, Bupleurum falcatum, Poria cocos, Glycyrrhiza uralensis, Cnidium officinale and Uncaria rhynchophylla. Yokukansan is used to treat insomnia and irritability as well as screaming attacks, sleep tremors and hypnic myoclonia, and neurological disorders which include dementia and Alzheimer's disease - the focus of this article. It is concluded that Yokukansan is a versatile herbal remedy with a variety of effects on various neurological states, without reported adverse effects. Traditional herbal medicines consist of a combination of constituents which account for the clinical effect seen. Likewise, the benefits of Yokukansan are probably attributable to the preparation as a whole, rather than to individual compounds.

In-vitro bioactivity of Venda medicinal plants used in the treatment of respiratory conditions.

Infectious diseases, especially those affecting the respiratory tract, represent a critical problem to health. Crude methanol and water extracts of 10 Venda plants reported to be used ethnomedically in the treatment of respiratory conditions were assessed for their antimicrobial activity against standard strains and clinical isolates of Candida albicans, Haemophilis influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium smegmatis using the disc diffusion assay. Four of the 10 plants tested possessed antimicrobial activity, but no activity against K. pneumoniae was observed. Minimum inhibitory concentrations, as determined by the broth microdilution assay, showed three plants, Securidaca longepedunculata, Syzygium cordatum, and Tabernaemontana elegans, to possess MICs

The ability of Warburgia salutaris extracts to protect against crystalline silica-induced cell injury.

In Southern Africa, the medicinal plant Warburgia salutaris is commonly used for the treatment of inflammatory and other diseases. The methanol extracts of W. salutaris were investigated with regard to a) production of proinflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma; b) activation of the transcription factor nuclear factor kappa B; and c) induction of deoxyribonucleic acid (DNA) damage and lipid peroxidation in the presence of crystalline silica particles. Due to its antioxidant properties, extracts of W. salutaris showed protective effects against crystalline silica-induced inflammatory cytokine expression, activation of nuclear transcription factor-kappaB, DNA strand breakage, and lipid peroxidation. Hence, W. salutaris may be a potential therapeutic agent against the fibrogenic and carcinogenic effects of crystalline silica.

Evaluation of Athrixia bush tea for cytotoxicity, antioxidant activity, caffeine content and presence of pyrrolizidine alkaloids.

In South Africa, Athrixia phylicoides DC. (bush tea) is widely used as a beverage, cough remedy and purgative. The commercialization of this tea in a similar vein to rooibos (Aspalathus linearis), is being considered. Traditional infusions and decoctions, as well as water and ethanol extracts, were prepared and screened. A related species, Athrixia elata Sond. (daisy tea), was included in many of the assays as a comparison. Extracts of Athrixia phylicoides and Athrixia elata were tested for toxic effects to brine shrimp larvae and the Vero kidney cell line. In both assays, the traditional preparations and aqueous extracts had little effect, but the ethanol extracts were relatively toxic. Antioxidant activity comparable to that found in rooibos was established. No detectable levels of caffeine were present in the Athrixia extracts following analysis using TLC and I/HCl spray reagent. Neither screening using spectrophotometry nor confirmation using gas chromatography-mass spectrometry analyses showed evidence of pyrrolizidine alkaloids in Athrixia phylicoides. Although a wider range of studies needs to be conducted prior to commercialization, these results support the development of bush tea as a healthy alternative to caffeine-containing beverages.

Pyrrolizidine alkaloids enhance alcohol-induced hepatocytotoxicity in vitro in normal human hepatocytes.

OBJECTIVE: Herbal remedies containing pyrrilidozine alkaloids (PA)s can induce liver damage, including hepato-sinusoidal obstruction syndrome (HSOS) or veno-occlusive liver disease (VOD). Some individuals misusing alcohol consume also teas and/or herbal remedies containing PA. The interaction or additive toxicity of alcohol to PA toxicity needs to be addressed. The objectives of this study are 1) to review the scientific literature on the PA-induced liver toxicity; 2) identify possible mechanism(s) involved in PA-induced hepatocytotoxicity in the presence or absence of ethanol (EtOH) in vitro in normal human hepatocytes (NHH) in primary culture. To respond to the first objective, we systematically search all the literature engines (PubMed, Google Scholar) for liver induced damage due to PAs and summarize the results in an introductory systematic review. ORIGINAL ARTICLE EXPERIMENTAL DESIGN AND METHODS: Cells were exposed to one dose of 100 mmol/L EtOH for 24 hrs and to 2 doses of 100 mmol/L EtOH for consecutive 24 hrs periods, in the presence or absence of PAs (10 mg/mL), or the caspase-3 inhibitor IDN-1965 (50 µmol/L). Cells were analyzed for apoptosis by light microscopy, immuno-histochemistry, measuring cytokeratin-18 fragmentation, and transmission electron microscopy (TEM) (6000 cells/treatment). Cytotoxicity was determined using succinate dehydrogenase (SDH) activity, an enzyme specific to the mitochondria. RESULTS: In NHH cells, a 100 mmol/L dose of Et-OH resulted in 22±2.5 apoptosis (p<0.001 vs. control). Two consecutive doses of 100 mmol/L Et-OH for 24 hrs each caused 36±3.0% apoptosis (p<0.001 vs. control and p<0.05 vs. one dose Et-OH). Pre-treatment with 50 µmol/L caspase inhibitor significantly reduced Et-OH-induced apoptosis [12±1.5% in 100 mmol/L (p<0.05) and 20±4.0% in 2×100 mmol/L (p<0.001)]. In addition, pre-treatment with 50 µmol caspase inhibitor in cells treated with PA + EtOH reduced apoptosis significantly (vs. non-exposed to caspase-inhibitor): Δ -22±3.0 % (p<0.05). HPC significantly decreased apoptosis compared to conditions lacking this supplementation in cells treated with EtOH-exposed cells present ballooning, Mallory bodies, changes in mitochondrial cristae and apoptosis by TEM. Pre-treatment with 50 µmol caspase inhibitor significantly reduced 100 mmol/L EtOH-induced (one dose) in NHH by 14±0.5% (p<0.05) compared to cells not exposed to the caspase-inhibitor. In cells treated concomitantly with PA and EtOH 100 mM Mallory-bodies and apo-necrotic cells have been observed. Pre-treatment with 50 µmol caspase inhibitor reduced the mitochondrial damage. A significant depletion in glutathione (GSH) was observed in Et-OH treated cells after 1 and 2 treatments (p<0.001 vs. control). Treatment with Et-OH enhanced PA-induced GSH-depletion and resulted in a significant increase in PA-induced cytotoxicity (p<0.001 vs. Et-untreated cells). Exposure to EtOH increased the cell culture media levels of the pro-inflammatory cytokine TNF. PA + EtOH-treated cells increased TNF-α levels in media compared to EtOH alone [86±8 vs. 53±5 pg/mL in cells exposed to 100 mmol/L EtOH (p<0.05) and 218±14 vs. 179±8 pg/mL in cells exposed to 2×100 mmol/L EtOH (p<0.05)]. CONCLUSIONS: PA up-regulates EtOH-induced hepatocytotoxicity by inducing the inflammatory cytokines and enhancing the apoptotic effects of ethanol. There is a need for monitoring herbal medicine in order to optimize traditional medicine use and maximize the clinical benefits. Additionally, there is necessary to communicate to physicians the possible negative results of herbal remedies use. Also, the interactions between herbal remedies and drugs of misuse should be communicated to consumers.

Studies on antibacterial, anti-inflammatory and antioxidant activity of herbal remedies used in the treatment of benign prostatic hyperplasia and prostatitis.

Crude water and ethanolic extracts of five herbal remedies reported in the literature for traditional treatment of benign prostatic hyperplasia (BPH) and/or prostatitis were investigated for their effect on hydroxyl scavenging activity, antibacterial activity and their ability to inhibit cyclooxygenase-1 and -2 (COX-1 and COX-2) catalysed prostaglandin biosynthesis. Both the water and ethanol extracts of Hypoxis hemerocallidea and Epilobium parviflorum inhibited the growth of Escherichia coli. All 10 extracts scavenged the hydroxyl radical but with various potencies (32-93%). Ethanolic extracts were the most active in inhibiting COX-1 catalysed prostaglandin biosynthesis. The ethanolic extract of Epilobium parviflorum showed inhibitory effects on both the COX-1 and -2 catalysed prostaglandin biosynthesis, inhibited growth of Escherichia coli and exerted antioxidant activity. Although these results support the traditional use of Epilobium parviflorum for treatment of prostatitis and BPH, further investigation is required, for this promising plant.

Fatalities involving illicit drug use in Pretoria, South Africa, for the period 2003 - 2012.

BACKGROUND: Globally, illicit drugs are responsible for many fatalities annually, yet accurate data on the nature and extent of these deaths in South Africa (SA) are lacking. OBJECTIVES: To investigate the presence and profile of illicit drugs detected in deceased persons who were subjected to medicolegal autopsies and upon whom analyses were carried out in search of illicit drugs in their body fluids at the Pretoria Medico-Legal Laboratory (PMLL), SA, over a 10-year period. METHODS: A retrospective descriptive case audit was conducted for the period 2003 - 2012. RESULTS: Screening for illicit drugs was requested in 385 out of 22 566 medicolegal autopsies. Results were available for only 281 of these cases, with 154 cases showing the presence of one or more illicit drugs. The demographic profile of positive cases indicated the majority to be male (90.3%) and white (85.1%). Decedents who tested positive for illicit drugs were predominantly aged between 20 and 30 years (51.9%). The most frequently detected drug was heroin, the presence of which was confirmed in 35.2% of cases, followed by cocaine in 19.9%. Alcohol in combination with an illicit drug or drugs was detected in 56 cases (36.4%). CONCLUSIONS: Results from this study indicate that illicit drugs were implicated in a considerable number of fatalities in Pretoria. However, it is believed that the figures are a gross under-representation of the actual number of drug users who died during this period. It is therefore recommended that further research be conducted and that drug screening be requested routinely when unnatural deaths are investigated at medicolegal mortuaries, not only to ensure the administration of justice but also to obtain more accurate data for purposes of public health programmes and improve insight into the burden of illicit drug use in SA.

Hepatic veno-occlusive disease as a result of a traditional remedy: confirmation of toxic pyrrolizidine alkaloids as the cause, using an in vitro technique.

BACKGROUND/AIMS: A child presented with hepatic veno-occlusive disease after having been administered a short course of treatment with a traditional herbal remedy. The child subsequently died. Postmortem liver histology confirmed the diagnosis. This study aimed to investigate the hypothesis that the herbal remedy was the cause of veno-occlusive disease. METHODS: Extracts of the traditional remedy were analysed by colorimetry and gas chromatography/mass spectrometry. Cultured hepatocytes were treated with an extract of the plant material and examined for morphological changes. RESULTS: The screening analyses indicated the presence of toxic pyrrolizidine alkaloids, which were later confirmed by gas chromatography/mass spectrometry. The cell studies indicated dose related toxicity, with necrosis at high concentrations and apoptosis and abnormalities of the cytoskeleton at lower concentrations. CONCLUSIONS: The simple screening techniques used allowed rapid confirmation of the presence of toxic pyrrolizidines in the remedy. The in vitro method confirmed the toxicity of herbal extracts to hepatocytes.

The toxicity of Callilepis laureola, a South African traditional herbal medicine.

OBJECTIVES: To review the literature on the toxicity of Callilepis laureola, and to assess the cytotoxicity of C. laureola in human hepatoblastoma Hep G2 cells in vitro. DESIGN AND METHODS: Cells were incubated for up to 48 h in the presence of increasing concentrations of an aqueous extract of C. laureola (0.3-13.3 mg/mL). Cytotoxicity was quantitated spectrophotometrically by the metabolism of the tetrazolium dye MTT. Cytoviability of the control cells was considered to be 100%. RESULTS: C. laureola produced cytotoxicity in a concentration-dependent manner. Cytotoxicity was significant at all concentrations tested (0.3-2.5 mg/mL, p < 0.05 vs. controls and 3.3-13.3 mg/mL, p < 0.0001 vs. controls). After 6 h, 100% toxicity was observed at a concentration of 6.7 mg/mL. CONCLUSION: C. laureola causes significant cytotoxicity in Hep G2 cells in vitro. These findings are in accordance with the observed hepatotoxicity in clinical cases of C. laureola poisoning.

A fatal case of pepper poisoning.

An 8 month old infant died as the result of administration of a traditional remedy, given orally as an infusion of a red powder. The symptoms on admission were coughing with diarrhoea and vomiting. Analysis of the powder using high-performance liquid chromatography (HPLC) confirmed the suspicion of capsaicin. Pepper preparations, which are used as traditional medicines, are highly toxic and their use on infants should be discouraged.

Metal concentrations in plants and urine from patients treated with traditional remedies.

A number of traditional South African herbal remedies which are associated with morbidity and mortality were analysed for selenium, manganese, copper, lead, zinc and mercury content. Few showed high levels of toxic metals, but in these the concentrations were sufficiently high as to cause concern since there is no quality control on the production of these remedies, which are the accepted form of medication for over 80% of the population. Urine samples, obtained from 65 patients admitted to hospital following treatment with a traditional herbal remedy were also analysed for metals. Only a small number of the patients had abnormally high levels of metal excretion. These data suggest that, in contrast to experience with traditional Chinese and Indian preparations, metal contamination from plants seems not to be a problem in traditional South African remedies.

Traditional herbal remedies used by South African women for gynaecological complaints.

Traditional remedies are part of the cultural and religious life of the African people. In this manuscript the nature and range of traditional remedies used for female complaints in relation to gynaecological conditions and disorders is reviewed. A total of 156 medicinal plant species are documented as being used for gynaecological complaints in South Africa. These are presented in a table with the local name, part of the plant used and specific gynaecological treatment. Medicinal plant species which are potentially toxic are noted as are the compounds responsible for the toxicity and the feature(s) of poisoning. Traditional remedies used in South Africa for the treatment of gynaecological problems are compared to those used elsewhere in the world. This manuscript indicates that a wide spectrum of herbal traditional remedies are used to regulate the menstrual cycle, enhance fertility and as either abortifacients or antiabortifacients.

Studies on antibacterial, antioxidant and fibroblast growth stimulation of wound healing remedies from South Africa.

Aqueous and methanol extracts of Urtica urens, Capparis tomentosa, Dicoma anomala, Leonotis leonorus, Xysmalobium undulatum, Helichrysum foetidum, Pterocarpus angolensis, Terminalia sericea and Gunnera perpensa, plants documented as being used for topical wound healing in the literature, were tested for antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa. Methanol and water extracts of two of these plants, Terminalia sericea and Gunnera perpensa, were more active compared to the other extracts against Streptococcus pyogenes and Staphylococcus aureus. The effects of the latter plants on fibroblast growth as well as oxidant production by N-formyl-methionyl-leucyl-phenylalanine were also studied. The water and methanol extracts of Terminalia sericea and Gunnera perpensa significantly decreased luciginin enhanced chemiluminescence at concentrations of 100 microg/ml and higher. However, the extracts had no effect on the growth of primary human fibroblasts.

The effect of Senecio latifolius a plant used as a South African traditional medicine, on a human hepatoma cell line.

A number of traditional remedies used in South Africa contain pyrrolizidine alkaloids, some of which are hepatotoxic. We investigated the effect on human HuH-7 cells of Senecio latifolius DC., a plant that is a component of some traditional remedies and which is known to contain toxic pyrrolizidine alkaloids. Cells were also treated with extracts of a standard pyrrolizidine, retrorsine. The changes in the gross morphology of the cells were studied using light microscopy after haematoxylin and eosin staining. The cytoskeleton was investigated using fluorescence-labelled anti-beta-tubulin antibody and the nuclear organisation was studied using fluorescence-labelled antinuclear antibodies. The plant extracts gave rise to dose-dependent gross morphological changes. At high doses, we observed necrosis and at lower doses, destruction of the cytoskeleton, nuclear fragmentation and apoptosis. Doses of less than the equivalent of 330 ng/ml retrorsine led to multinucleated cells with failure in spindle formation and clumping of nuclear chromatin. This latter finding suggests that chronic low-dose treatment with such traditional remedies could give rise to teratogenic and/or carcinogenic effects.

Detection of poisoning by Impila (Callilepis laureola) in a mother and child.

Poisoning with impila (Callilepis laureola) is a recurring phenomenon in South Africa. Cases of poisoning with other plants which contain atractyloside also occur in Europe and the Americas. Since poisoning leads to rapid death from renal and/or hepatic failure, it is suspected that many cases are undiagnosed; this is especially so in South Africa, where patients may die without reaching hospital and do not often admit to ingestion of a traditional remedy. We have developed a thin layer chromatographic method for the detection of impila constituents in urine. We describe the clinical symptoms and the application of the screening method to diagnosis in the case of a mother and child, who both showed symptoms of impila poisoning; the mother died but the child survived. This method is rapid and may be used for the definitive diagnosis in cases of poisoning with atractyloside-containing plants.

The cytotoxic effects of a traditional Zulu remedy, impila (Callilepis laureola).

The traditional Zulu remedy impila (Callilepis laureola) can cause acute fatal hepatocellular necrosis, especially in children. We investigated the mechanism(s) of toxicity using HuH-7 hepatocytes. Impila tubers were extracted with boiling water and the aqueous extract was used at different concentrations to study the effects on the morphology of the cells. Flow cytometry and labelling with fluorescent antibodies to tubulin were also used. At high concentrations, necrosis occurred; however, at lower concentrations, the extracts gave rise to a variety of changes including hypercondensation of chromatin, multinucleate cells, nuclear fragmentation and apoptosis. In addition, we observed destruction of cytoplasmic tubulin. These findings give further insight into the mechanism of toxicity of herbal remedies containing atractyloside.

Adverse effects associated with the use of South African traditional folk remedies.

UNLABELLED: Although the toxicity of traditional folk remedies is well known in Africa, it is a subject which is surrounded by secrecy and has not been comprehensively studied. OBJECTIVES: The aims of this study are to describe the clinical features of patients admitted to hospital with a confirmed history of using folk remedies, and to gather data on their toxicity in a systematic fashion. DESIGN: Prospective case series. SETTING: Paediatric and adult wards of academic hospitals in Johannesburg, South Africa. SUBJECTS: The study population included 103 patients ranging from one day to 75 years of age, all of whom had recent folk remedy use. MAIN OUTCOME MEASURES: All available clinical data were analysed. Primary outcomes were the presence of renal and liver dysfunction, death or discharge from hospital. RESULTS: The most common clinical features on presentation were dehydration (51%), vomiting (46%), jaundice (40%), diarrhoea (39%), altered mental status (37%) and oligoanuria (30%). Renal dysfunction was present in 76% of patients and liver dysfunction in 48%. The overall mortality was 34%. The odds ratio of death was 5.1 (95% CI 1.41 to 18.5) in patients with renal dysfunction (p = 0.0077) and 5.35 (95% CI 1.99 to 14.4) in patients with liver dysfunction (p = 0.0006). CONCLUSION: Renal and liver dysfunction are frequently associated with use of folk remedies, and mortality in these patients is high. In view of the large numbers of African individuals living in the United States and Europe, it is important for physicians elsewhere to be aware of the potential toxicity of African folk remedies, and to inquire about their use.

In vitro effect of N-acetylcysteine on hepatocyte injury caused by dichlorodiphenyltrichloroethane and its metabolites.

The organochlorine pesticide, dichlorodiphenyltrichloroethane (DDT), is still used to combat the spread of malaria in several developing countries despite its accumulation and known hepatotoxic effects that have been demonstrated both in vitro and in vivo. N-Acetylcysteine (NAC) is a recognized hepatoprotective agent that has been reported to reduce hepatotoxicity initiated by many different compounds. The aim of this study was to determine whether NAC could counter in vitro hepatocyte injury induced by DDT or its two major metabolites, dichlorodiphenyldichloroethylene and dichlorodiphenyldichloroethane. HepG2 cell cultures were used to assess the following parameters of toxicity: cellular viability, intracellular levels of reactive oxygen species (ROS), mitochondrial membrane potential and initiation of apoptosis. None of the three test compounds induced ROS generation, yet exposure to any of the three compounds produced mitochondrial hyperpolarization, which was countered by NAC pretreatment. All three test compounds also induced apoptotic cell death, which was inhibited by NAC. Despite NAC counteracting some adverse intracellular changes due to organochlorine exposure, it appeared to aggravate the cytotoxic effects of the organochlorine compounds at low test concentrations. As the same outcome may also occur in vivo, results from the present study raise concern about the use of NAC as treatment for DDT-induced hepatotoxicity.

Effect of an alkaloidal fraction of Tabernaemontana elegans (Stapf.) on selected micro-organisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Bacterial infections remain a significant threat to human health. Due to the emergence of widespread antibiotic resistance, development of novel antibiotics is required in order to ensure that effective treatment remains available. There are several reports on the ethnomedical use of Tabernaemontana elegans pertaining to antibacterial activity. AIM OF THE STUDY: The aim of this study was to isolate and identify the fraction responsible for the antimicrobial activity in Tabernaemontana elegans (Stapf.) root extracts. MATERIALS AND METHODS: The active fraction was characterized by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). Antibacterial activity was determined using the broth micro-dilution assay and antimycobacterial activity using the BACTEC radiometric assay. Cytotoxicity of the crude extract and fractions was assessed against primary cell cultures; lymphocytes and fibroblasts; as well as a hepatocarcinoma (HepG2) and macrophage (THP-1) cell line using the Neutral Red uptake and MTT assays. RESULTS: The crude root extracts were found to contain a high concentration of alkaloids (1.2%, w/w). GC-MS analysis identified the indole alkaloids, voacangine and dregamine, as major components. Antibacterial activity was limited to the Gram-positive bacteria and Mycobacterium species, with MIC values in the range of 64-256µg/ml. When combined with antibiotics, additive antibacterial effects were observed. Marked cytotoxicity to all cell lines tested was evident in the MTT and Neutral Red uptake assays, with IC(50) values <9.81µg/ml. CONCLUSIONS: This study confirms the antibacterial activity of Tabernaemontana elegans and supports its potential for being investigated further for the development of a novel antibacterial compound.