Winston A. Morgan

The ethanol extract of Scutellaria baicalensis and the active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells

Despite a lack of scientific authentication, Scutellaria baicalensis is clinically used in Chinese medicine as a traditional adjuvant to chemotherapy of lung cancer. In this study, cytotoxicity assays demonstrated that crude ethanolic extracts of S. baicalensis were selectively toxic to human lung cancer cell lines A549, SK-LU-1 and SK-MES-1 compared with normal human lung fibroblasts. The active compounds baicalin, baicalein and wogonin did not exhibit such selectivity. Following exposure to the crude extracts, cellular protein expression in the cancer cell lines was assessed using 2D gel electrophoresis coupled with MALDI-TOF-MS/Protein Fingerprinting. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity. These observations were supported by PI staining cell cycle analysis using flow cytometry and Annexin-V apoptotic analysis by fluorescence microscopy of cancer cells treated with the crude extract and pure active compounds. Moreover, specific immunoblotting identification showed the decreased expression of cyclin A results in the S phase arrest of A549 whereas the G(0)/G(1) phase arrest in SK-MES-1 cells results from the decreased expression of cyclin D1. Following treatment, increased expression in the cancer cells of key proteins related to the enhancement of apoptosis was observed for p53 and Bax. These results provide further insight into the molecular mechanisms underlying the clinical use of this herb as an adjuvant to lung cancer therapy.

Ethnopharmacological survey and in vitro evaluation of wound-healing plants used in South-western Nigeria.

ETHNOPHARMACOLOGICAL RELEVANCE Traditional healers in Nigeria employ a range of plant preparations as wound healing agents. Despite the use of local plants in wound healing, there is only scant literature on the wound healing properties of these plants to support the continued therapeutic application of these herbal remedies. AIM OF THE STUDY To document plants commonly used to treat wounds in South-western Nigeria and to test the scientific basis of such claims using relevant in vitro tests. MATERIALS AND METHODS Structured questionnaires were used to determine which plant preparations are in common use, via interviews with Yoruba traditional healers. Aqueous and ethanolic extracts of the nine most common plants cited by the healers were collected, identified and tested using relevant in vitro wound healing assays. Minimum inhibitory concentrations (MIC) were determined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Antioxidant activity was measured by DPPH assay and fibroblast proliferation determined by neutral red assay. RESULTS A total of 20 traditional healers from South-western Nigeria were involved in the study. Thirty-six plant species were recorded with their local names and parts used in the traditional wound healing preparations. Ethanolic extracts of nine species most frequently cited by the healers exhibited strong antioxidant activities (3.8-31.3 μg/ml) comparable to ascorbic acid (7.3 μg/ml). Crude extracts of the selected plants also inhibited the growth of bacteria with MIC values 0.3-7.6 mg/ml. Ethanol extracts of Bridelia ferruginea Benth. (1-30 μg/ml) and Parkia biglobosa Jacq. (15-30 μg/ml) influenced the proliferation of dermal fibroblasts significantly (p<0.05). Extracts from the remaining seven plants either had no effect on fibroblast proliferation or were cytotoxic. CONCLUSION Traditional use of many wound-healing plants from Nigeria can be rationalised by activity determined in relevant in vitro investigations of ethanol and aqueous extracts. These results support the traditional selection of these plants in South-western Nigeria for wound healing.

Methapyrilene hepatotoxicity is associated with oxidative stress, mitochondrial disfunction and is prevented by the Ca2+ channel blocker verapamil.

Methapyrilene (MP) is an unusual hepatotoxin in that it causes periportal necrosis in rats. The mechanism of acute methapyrilene hepatotoxicity has, therefore, been investigated in cultured male rat hepatocytes. Addition of methapyrilene to rat hepatocytes resulted in a time- and dose-dependent loss in cell viability between 4 and 8 h of incubation as judged by cellular enzyme leakage. The cytochrome P450 (CYP) inhibitor metyrapone protected against methapyrilene-mediated toxicity suggesting that MP is metabolised by CYP for toxicity. The concentration-dependent protection from methapyrilene toxicity afforded by metyrapone correlated with an inhibition of microsomal CYP2C11-associated androstenedione 16alpha hydroxylase activity, and hepatocytes prepared from hypophysectomised rats (containing reduced levels of microsomal immunodetectable CYP2C11 and associated androstenedione 16alpha hydroxylase activity) showed resistance to the toxic effects of methapyrilene. These data suggest that the toxicity of methapyrilene is predominantly dependent on the CYP2C11 isoform. Treatment of hepatocytes with a toxic concentration of MP caused oxidative stress as indicated by increases in NADP+ levels within 2 h and cellular thiol oxidation as evidenced by a reduction--but not complete loss--in glutathione levels. Methapyrilene hepatotoxicity was associated with an early loss in mitochondrial function, as indicated by mitochondrial swelling and significant losses in cellular ATP within 2 h. Co-incubation of methapyrilene-treated hepatocytes with inhibitors of inner mitochondrial transition permeability pore opening--cyclosporin A or the thiol reductant dithiothreitol--abrogated cell death suggesting that pore opening and loss of mitochondrial Ca2+ homeostasis play a significant role in methapyrilene-mediated cell death. Co-incubation of methapyrilene-treated hepatocytes with the phenylalkylamine calcium channel blocker verapamil--but not by treating cells in a nominally calcium-free medium--also abrogated cell death, suggesting that if Ca2+ is involved in cell killing then it is dependent on an intracellular Ca2+ pool. Pre-treatment of hepatocytes for 1 h with verapamil--to inhibit intracellular Ca2+ pool filling--increased the potency of verapamil protection against methapyrilene toxicity by approximately 100-fold. Taken together, these data indicate that methapyrilene intoxication leads to mitochondrial disfunction and suggest a critical role for a loss of mitochondrial Ca2+ homeostasis in this model of hepatocyte death.

The role of reactive oxygen species in adriamycin and menadione-induced glomerular toxicity.

Redox cycling leading to oxidative stress has been proposed as the mechanism by which adriamycin induces glomerular toxicity in rats. The present study compares the extent of the oxidative stress and cytotoxicity induced by adriamycin to menadione (a model redox cycling quinone) in freshly isolated rat glomeruli. Adriamycin and menadione (25 microM) decreased de novo protein synthesis (measured by 3H-proline incorporation into acid-precipitable glomerular protein) by 50 and 85%, respectively, in 2 h. By contrast, menadione at 25 microM reduce glomerular membrane integrity (as assessed by lactate dehydrogenase leakage), adriamycin reduced membrane integrity at 500 microM adriamycin. Reactive oxygen species (ROS) were measured by the oxidation of dihydrodichlorofluorescein. Menadione (25 microM) and adriamycin (25 microM) increased ROS formation to 260 and 156% of controls after 30 min incubation, respectively. Oxidative stress was assessed by measuring the intracellular level of reduced glutathione (GSH) and the decrease of the NADPH/NADP- ratio which stimulates the pentose phosphate pathway (PPP): (a) menadione (25-100 microM) reduced glomerular GSH to 10-20% of controls, adriamycin (25-100 microM) had no effect; (b) menadione (10 microM) increased PPP activity 6-fold, while adriamycin (125 microM) had only a 2-fold effect. Although adriamycin and menadione generate extensive ROS and decrease protein synthesis, there was no correlation between the extent of oxidative stress and cytotoxicity in glomeruli exposed to adriamycin. These results suggest that oxidative stress may not be the primary mechanisms by which adriamycin induces selective glomerular toxicity.

Are Bile Acids Involved in the Renal Dysfunction of Obstructive Jaundice? An Experimental Study in Bile Duct Ligated Rats

Background. Surgery on patients with obstructive jaundice is associated with a significant risk of postoperative renal failure. Bile acids are implicated as nephrotoxins because they accumulate in the plasma and the kidney becomes their only excretory route in cholestasis. The experimental evidence favoring this proposal is inadequate and unconvincing. Therefore, we designed an animal experiment involving bile duct ligated (BDL) rats in which we could correlate variations in serum and urine bile acids with indices of nephrotoxicity and renal function. Hypothesis. Bile acids are putative nephrotoxins. Materials and Methods. Total serum and urine bile acid concentrations and profiles were determined using liquid chromatography/gas chromatography/mass spectrometry selected ion monitoring. Nephrotoxicity was assessed by renal histopathology and by determination of the urinary activities of the following enzymes: muramidase, glutamate dehydrogenase, alkaline phosphatase, N‐acetyl‐β‐D‐glucosaminidase, and lactate dehydrogenase. Renal function was assessed by measuring urine osmolality, daily osmolar excretion, sodium excretion (UNaV), potassium excretion (UKV), and total protein and albumin excretion. Results. Maximum plasma concentrations and renal clearance of bile acids occurred between the third or fourth postoperative day following BDL. This peak coincided with maximal disruption of proximal convoluted tubule architecture and postoperative changes in renal function—increased urine flow rate and decreases in urine osmolality and sodium excretion. Thereafter, 1) plasma levels of bile acids returned toward normal levels, 2) urinary bile acid clearance declined, 3) normal renal histology was restored, and 4) normal renal function was reestablished. Throughout this period, fluctuations in enzymuria were evident. However, these shifts did not coincide with plasma and urine bile acid concentrations and histological and functional changes. Discussion and Conclusions. Transient functional impairment of renal cation and water transport and nonspecific morphological changes in the proximal convoluted tubule occur 3 to 4 days following bile duct ligation in rats. These functional and morphological changes occurred when plasma total and urinary bile acids were at their peaks. Although it is tempting to equate association with causality, we cannot implicate bile acids as being responsible for the aberrations in renal function and structure following BDL. Accordingly, we have concluded that elevated plasma concentrations of bile acids are renal exacerbates acting in concert with other factors, be they prerenal or renal in origin to precipitate a cascade of events leading to postoperative renal failure in cholestasis.

Antibacterial, antioxidant and fibroblast growth stimulation activity of crude extracts of Bridelia ferruginea leaf, a wound-healing plant of Nigeria

AIM OF THE STUDY Determination of pharmacological activity relevant to wound healing of Bridelia ferruginea leaf, a traditional medicine used to treat wounds in rural Nigeria. MATERIALS AND METHODS Aqueous and ethanolic leaf extracts were tested against bacterial species of relevance to wound infections: Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The ethanolic extracts were assessed for their ability to stimulate the growth of human dermal fibroblasts (FS5) and protect against damage induced by hydrogen peroxide. Antioxidant activity was also assessed using the DPPH assay. RESULTS Both aqueous and ethanolic extracts had weak antibacterial activity (MIC>470 μg/ml). A significant effect (p<0.001) on the growth of FS5 fibroblasts was observed only at a concentration of 5 μg/ml (28% increase), above which the extracts appeared toxic to the cells. The ethanolic extract offered the highest protection against H(2)O(2) damage to FS5 cells, comparable with catalase (82% at 250 μg/ml). The DPPH assay revealed antioxidant activity of the ethanolic leaf extract with IC(50) 12.5±0.3 μg/ml comparable to l-ascorbic acid (7.3±0.1 μg/ml). CONCLUSION The antibacterial, modest fibroblast stimulation activity and relatively strong antioxidant activity lend some support to the topical use of Bridelia ferruginea leaf for wound-healing in the traditional medicine of South-western Nigeria.

A biochemical and NMR spectroscopic study of hydrazine in the isolated rat hepatocyte

Using isolated rat hepatocytes the biochemical effects of hydrazine have been investigated using both conventional assay techniques and high resolution proton NMR. High resolution proton NMR revealed that hydrazine caused a significant increase in alanine and lactate levels in the incubation buffer, whereas levels of β-hydroxybutyrate were decreased. NMR also detected metabolites of hydrazine notably acetylhydrazine and a cyclised hydrazone formed with α-ketoglutarate. Changes were detected in NADH and NADPH, ATP, succinate dehydrogenase (SDH) and total non-protein sulphydryl groups (TNPSH). However, the changes in pyridine nucleotides occurred at higher concentrations than those affecting succinate dehydrogenase and ATP. Similarly, the depletion of TNPSH occurred at a higher concentration and with a different time course to that seen with ATP depletion and inhibition of succinate dehydrogenase.

Quinone-induced DNA single strand breaks in rat hepatocytes and human chronic myelogenous leukaemic K562 cells

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.

RETRACTED: Antibacterial activity and in vitro cytotoxicity of extracts and fractions of Parkia biglobosa (Jacq.) Benth. stem bark and Ageratum conyzoides Linn. leaves

Many species of plants in African countries are widely used in the rural communities where there is little or no access to modern medicine. However, the safety and effectiveness of these medicinal plants are poorly evaluated. The stem bark of Parkia biglobosa Jacq. and leaves of Ageratum conyzoides Linn. were investigated for their antibacterial and cytotoxic activities. The plant materials were extracted with 95% ethanol, and fractionated with petroleum ether, chloroform and ethyl acetate. The antibacterial effects of the extracts and fractions of the plant materials were assayed on the bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA) and Clostridium perfringes. Ethanol extracts of P. biglobosa and A. conyzoides were screened for cytotoxicity using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Two cancer cell lines (SK-MES 1 and SK-LU 1) and one normal cell line (human skin fibroblast cell line, FS5) were used for the screening of the extracts and the fractions obtained. The ethanolic extracts and fractions of P. biglobosa and A. conyzoides showed the best activity against E. coli, S. aureus and MRSA. All fractions of A. conyzoides leaves have no activity against P. aeruginosa. Human lung cancer cell lines (SK-LU 1 and SK-MES 1) and human skin fibroblast cell line (FS5 cells) were treated with various concentrations (3.9μg/ml-2mg/ml) of the extracts and fractions for 24h. SK-MES 1 cells are more susceptible to treatment with the plant fractions. All the fractions of A. conyzoides leaves and the petroleum ether fraction of P. biglobosa were cytotoxic to SK-MES 1 cells, which to some extent may support their traditional inclusion in herbal preparations for treatment of cancer. The overall results provided evidence that the studied plant extracts might be potential sources of new antibacterial and anticancer drug.

The relationship between sodium chloride concentration and bile acid cytotoxicity in cultured kidney cells

Patients with obstructive jaundice suffer an increased incidence of mortality from post operative renal failure, which may be related to elevated circulating bile salts. This study assesses the effects of increased ionic strength (similar to that found in the kidney inner medulla) on bile salt critical micellar concentration (CMC) and cytotoxicity to renal medullary epithelial primary cultures and MDCK and NRK cell lines representing the distal and proximal tubular cells respectively. The CMC of chenodeoxycholic acid decreased from 2.86 +/- 0.07 (in isotonic Earles Hepes buffer) to 2.30 +/- 0.07, 1.99 +/- 0.09 and 1.46 +/- 0.08 mM following the addition of 150, 250 and 500 mM NaCl. Similarly, the CMC of deoxycholic acid was reduced from 3.18 +/- 0.1 to 2.84 +/- 0.1, 2.26 +/- 0.1 and 1.79 +/- 0.09 mM by the addition of 150, 250 and 500 mM NaCl. Increasing the ionic strength of the culture medium of medullary epithelial cells by the addition of 150 mM NaCl, decreased viability by 39% (p < 0.01), 24% (p < 0.001) and 40% (p < 0.001) for lithocholic (25 microM), chenodeoxycholic (100 microM) and deoxycholic acids (100 microM), respectively. A similar increase in the ionic strength of the culture medium of MDCK cells decreased viability by 79% (p < 0.01), 46% (p < 0.01) and 15% (p < 0.01) for lithocholic (15 microM), chenodeoxycholic (100 microM) and deoxycholic (50 microM), respectively. Adding 200 mM urea to medium supplemented with 150 mM NaCl (to further increase osmolality but not ionic strength) had no effect on the cytotoxicity bile salts in MDCK cells. The addition of 150 mM NaCl to the culture medium of NRK cells resulted in a decrease viability of 15% (p < 0.01), 27% (p < 0.01) and 60% (p < 0.01) following exposure to either lithocholic (15 microM), chenodeoxycholic (50 microM) or deoxycholic acids (50 microM) respectively. These results show that increasing NaCl concentrations lowers CMC of bile salts and increase cytotoxicity in medullary epithelial primary, MDCK and NRK cells. This suggests that the high NaCl levels in the kidney inner medulla would reduce bile salt CMC such that they could damaged renal cells. This may, in part, explain the increased susceptibility of the kidney during obstructive liver disease.