Inhibition of mitochondrial function: An alternative explanation for the antipyretic and hypothermic actions of acetaminophen.

AIMS: Acetaminophen is the medication of choice when treating fever because of its limited anti-inflammatory effects. However at overdose it can cause mitochondrial dysfunction and damage, often associated with metabolism to N-acetyl-p-benzoquinone imine (NAPQI). What has never been investigated is whether the inhibition of mitochondrial function, particularly fatty acid uptake and oxidation could be the key to its antipyretic and hypothermic properties. METHODS: Mitochondrial function and fatty acid oxidation (FAO) was determined by measuring oxygen consumption rate (OCR) in isolated mitochondria and in 3T3-L1 adipocytes using the XFp Analyser. Basal fatty acids and adrenergic stimulated OCR of mitochondria and 3T3-L1 adipocytes were assessed with acetaminophen and compared to NAPQI, etomoxir, and various mitochondrial stress compounds. KEY FINDINGS: Using the XFp Analyser, acetaminophen (10 mM) decreased FAO by 31 % and 29 % in basal and palmitate stimulated adipocytes. NAPQI (50 µM) caused a 63 % decrease in both basal and palmitate stimulated FAO. Acetaminophen (10 mM) caused a 34 % reduction in basal and adrenergic stimulated OCR. In addition acetaminophen also inhibited complex I and II activity at 5 mM. NAPQI was far more potent at reducing mitochondrial respiratory capacity, maximum respiratory rates and ATP production than acetaminophen. SIGNIFICANCE: These studies demonstrate the direct inhibition of mitochondrial function by acetaminophen at concentrations which have been shown to reduce fever and hypothermia in mammals. Understanding how antipyretics directly affect mitochondrial function and heat generation could lead to the development of new antipyretics which are not compromised by the anti-inflammatory and toxicity of the current medications.

Inhibition of lipolysis: A novel explanation for the hypothermic actions of acetaminophen in non-febrile rodents.

Acetaminophen is both widely used to treat children with fever and is also responsible for thousands being hospitalised annually. Historically the antipyretic actions of acetaminophen were attributed to the inhibition of cyclooxygenase (COX-1/2) enzymes and more recently a novel COX-1 variant (COX-3) located in the brain. However, the evidence for acetaminophen-mediated COX inhibition remains contentious. This study assesses the impact of acetaminophen and other putative COX-3 inhibitors on the release of fatty acids during lipolysis as an alternative mechanism by which antipyretics can reduce body temperature during fever. 3T3-L1 adipocytes, primary brown adipocytes and isolated mitochondria were exposed to COX-3 inhibitors and lipolysis and mitochondrial electron transport chain function assessed. Acetaminophen, aminopyrine and antipyrine at 1-10 mM caused a significant decrease (up to 70%; P < 0.01, from control) in lipolysis within 1, 3 and 24 h without affecting cell viability. The inhibition was observed regardless of where along its signalling pathway lipolysis was stimulated. All three compounds were found to significantly attenuate mitochondrial function by up to 30% for complex I and 40% for complex II (P < 0.01, from control). These novel observations combined with the known limited inhibition of the COX enzymes by acetaminophen suggest both the antipyretic and hypothermia induced by acetaminophen and related compounds could be attributed to the direct inhibition of lipolysis and mitochondrial function, rather than cyclooxygenase inhibition centrally. Further these observations could provide new drug targets for reducing fever with the added bonus of fewer individuals being hospitalized by accidental acetaminophen overdose.

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 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.

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.

The use of High Performance Thin-Layer Chromatography to determine the role of membrane lipid composition in bile salt-induced kidney cell damage.

INTRODUCTION: In obstructive liver disease bile salts are known to accumulate in and damage specific kidney cells. High Performance Thin-Layer Chromatography (HPTLC) was used to determine the membrane lipid composition of a range of kidney cells. METHODS: Kidney cells were exposed to three hydrophobic bile salts (lithocholic, deoxycholic and chenodeoxycholic acids) and cytotoxicity was determined. In addition membrane lipids from the cells were extracted in a chloroform:methanol (2:1, v/v) solution and quantified by HPTLC. RESULTS: The results reveal a differential toxicity to the bile acids with IC(50) values ranging from 79+/-5 microM to 394+/-13 microM. When the lipid composition of the most and least susceptible cells were assayed, the least susceptible cells had a much higher lipid composition (46.6+/-3.7 microg/mg protein compared to 28.1+/-5.2 microg/mg protein for the least susceptible cells). DISCUSSION: These results suggest that HPTLC may be a useful technique when determining the mechanisms of toxicity of compounds which cause the disruption of the cell membrane.

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-beta-D-glucosaminidase, and lactate dehydrogenase. Renal function was assessed by measuring urine osmolality, daily osmolar excretion, sodium excretion (U(Na)V), potassium excretion (U(K)V), 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.