Leaching of pesticides in tea brew.

A cup of tea that cheers can also be an important route of human exposure to pesticide residues. It is important to evaluate the percent transfer of pesticide residue from dried (made) tea to tea infusion, as tea is subjected to an infusion process prior to human consumption. To investigate the pesticide translocation, 13 pesticides commonly used on tea were studied by subjection of fortified teas to infusion. Analytes of interest were quantified by gas-liquid chromatography with nitrogen-phosphorus and electron capture detectors. Interestingly, water solubility of pesticides did not necessarily indicate a shift of residues toward their preferential accumulation in infusion. The pesticides with larger partition coefficient (K(ow)) values remained nonextractable in infusing water. Further, boiling for longer periods (extended brewing time) resulted in higher transfer of pesticides to tea brew.

A modified extraction and clean-up procedure for the detection and determination of parathion-methyl and chlorpyrifos residues in tea.

Recent advances in methodology and instrumentation have made possible the detection and determination of pesticides at microgram kg-1 (ppb) levels. The sensitivity of a method of analysis depends greatly on the efficient extraction of the pesticide and the subsequent clean-up of the extract. The extract from green tea leaves is a mixture of aroma components, polyphenols and caffeine. The preparation of made tea from green tea leaves adds to this complexity by concentrating these coextractives. Conventional clean-up techniques provide poor recoveries for parathion-methyl and chlorpyrifos from both green tea leaves and made tea. This arises from interference by caffeine during gas chromatography, as it has a similar retention time to the two pesticides and peaks overlap. A modification to the protocol based on a solvent partitioning process using dichloromethane and subsequent washing of the extracts with warm water removed the caffeine, and pigments were removed by column chromatography. Recoveries ranging from 80 to 90% were then obtained for both pesticides.

Phenylenediamine induced hepatocyte cytotoxicity redox. Cycling mediated oxidative stress without oxygen activation.

Muscle necrosis induced by various phenylenediamine derivatives has been correlated with their autoxidation rate. However, a more detailed investigation of the cytotoxic mechanism using a model system of isolated hepatocytes and 2,3,5,6-tetramethylphenylenediamine (DD) shows little oxygen activation as indicated by the absence of cyanide resistant respiration, lipid peroxidation and lack of cytoprotection by iron chelators, superoxide dismutase mimics and xanthine oxidase inhibitors. Cytotoxicity was however attributed to oxidative stress as GSH was not only rapidly oxidized to GSSG but mixed protein disulfide formation also occurred. Furthermore, the disulfide reductant dithiothreitol added some time after DD restored protein thiols and prevented further cytotoxicity. This oxidative stress was attributed to a futile two electron redox cycle involving oxidation of DD to the corresponding diimine by the mitochondrial electron transport chain and rereduction by DT diaphorase. Evidence suggesting this was that both diimine accumulation and the ensuing cytotoxicity were markedly increased by inactivating hepatocyte DT diaphorase but were prevented by a subtoxic concentration of the mitochondrial respiratory inhibitor cyanide. Furthermore, addition of NADH generating substrates such as lactate, sorbitol, xylitol or ethanol prevented DD induced GSH oxidation and cytotoxicity. This suggests that DD undergoes intracellular redox cycling without oxygen activation until the hepatocyte is unable to maintain redox homeostasis and mixed protein disulfide cytotoxicity ensues.

2-Chloroacetaldehyde-induced cerebral glutathione depletion and neurotoxicity.

2-Chloroacetaldehyde (CAA) formed during the metabolism of the anti-cancer drug ifosfamide (IP) has been implicated in ifosfamide-related neurotoxicity during chemotherapy but the neurotoxic mechanisms are unknown. We have found that IP (900 mg kg-1, p.o.) caused lethargy and mild hind limb paralysis after 6 h. Neurotoxicity and IP-induced mortality was markedly enhanced in mice pretreated with either phenobarbital or dexamethasone to induce cytochrome P4503A. Cerebral glutathione (GSH) levels were also markedly depleted in these pretreated mice. 2-Chloroethanol (92 mg kg-1, i.p.) (CE) also caused a 50% reduction in cerebral GSH 6 h after administration to mice. At this time maximum lethargy and unresponsiveness to touch was apparent in CE-treated mice. Severe hind limb paralysis developed and death ensued 12-18 h later. Prior depletion of cerebral GSH with 2-cyclohexene-1-one greatly accelerated the onset of CE-induced neurotoxicity suggesting that cerebral GSH status is an important determinant of CE-induced neurotoxicity. Furthermore, pretreatment with N-acetylcysteine delayed both CE-induced neurotoxicity and cerebral GSH depletion. Induction of cerebral but not hepatic CYP2E1 by ethanol before CE challenge also potentiated CE-induced cerebral GSH depletion and neurotoxicity. Hepatic GSH depletion was unaffected suggesting that CE-induced paralysis is dependent on a cerebral but not a hepatic CYP2E1 catalysed oxidation of CE to CAA. Ethanol was neuroprotective even if given 60 min after CE and prevented further cerebral GSH depletion. 4-Methylpyrazole, a CYP2E1 and alcohol dehydrogenase inhibitor, prevented both CE-induced hepatic and cerebral GSH depletion and paralysis. This suggests that the neurotoxicity associated with IP chemotherapy involves activation of chloroethanol by cerebral CYP2E1 to chloroacetaldehyde which mediates cerebral GSH depletion. Neurotoxicity may be prevented by restoring cerebral GSH status and/or by preventing activation of CE by CYP2E1 with ethanol.

The involvement of cytochrome P4502E1 in 2-bromoethanol-induced hepatocyte cytotoxicity.

The cytotoxicity of 2-bromoethanol towards hepatocytes isolated from rats was concentration-dependent (EC(50)100 mu M, 2 hr). Bromoacetaldehyde was more toxic (EC(50)60 mu M, 2 hr) and bromoacetic acid was less toxic (EC(50)150 mu M, 2 hr). Glutathione (GSH) depletion occurred before cytotoxicity ensued and GSH depleted hepatocytes were more susceptible to 2-bromoethanol. Lipid peroxidation increased steadily 1 hr after 2-bromoethanol addition and antioxidants, iron chelators or hypoxia prevented 2-bromoethanol induced lipid peroxidation and cell lysis. Alcohol dehydrogenase inhibitors, methyl pyrazole or dimethyl sulfoxide only partly prevented 2-bromoethanol induced GSH depletion, lipid peroxidation and cytotoxicity. However, cytochrome P4502E1 (CYP2E1) inhibitors/substrates were more effective at preventing 2-bromoethanol-induced GSH depletion, lipid peroxidation and cytotoxicity suggesting that 2-bromoethanol is mostly metabolically activated by CYP2E1. Also, hepatocytes isolated from CYP2E1 induced rats were more susceptible to 2-bromoethanol and hepatocytes isolated from rats pretreated with carbon disulfide to inactivate CYP2E1 were more resistant to 2-bromoethanol treatment. Formation of S-(formylmethyl)glutathione during 2-bromoethanol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by cytochrome P4502E1 inhibitors/substrates or by Anti-Rat CYP2E1. Furthermore, aldehyde dehydrogenase inhibitors-cyanamide or chloral hydrate increased 2-bromoethanol dependent hepatocyte susceptibility. This suggests that 2-bromoethanol is preferably metabolised by CYP2E1 dependent monoxygenase to form 2-bromoacetaldehyde which causes cell lysis as a result of GSH depletion and lipid peroxidation.

Prevention of cyanide-induced cytotoxicity by nutrients in isolated rat hepatocytes.

The effects of various glycolytic substrates and keto acid metabolites on the cytotoxic effects of cyanide have been studied with isolated rat hepatocytes. The sequence of cytotoxic events with 2 mM cyanide was an immediate inhibition of respiration followed by ATP depletion. Disruption of the plasma membrane occurred when 85-90% of ATP levels had been depleted. Fructose, dihydroxyacetone, glyceraldehyde, pyruvate, and alpha-ketoglutarate prevented cyanide-induced cytotoxicity and ATP depletion. Hepatocyte respiration was also restored by all except fructose. Fructose, unlike the others, also did not prevent cytotoxicity if added 30-60 min after cyanide. Fluoride, an inhibitor of the glycolytic enzyme enolase, prevented protection by fructose but not dihydroxyacetone or glyceraldehyde, suggesting that dihydroxyacetone and glyceraldehyde are cytoprotective by trapping cyanide, thereby restoring cytochrome oxidase activity and cellular ATP levels. Fructose, on the other hand, may be cytoprotective by supplying ATP through glycolysis. Hepatocytes isolated from fasted rats were five- to sevenfold more susceptible to cyanide-induced cytotoxicity. Furthermore, all glycogenic and gluconeogenic amino acids and carbohydrates were cytoprotective against cyanide toxicity toward fasted hepatocytes, suggesting that cellular energy stores determine their resistance to cyanide.

Chloroacetaldehyde-induced hepatocyte cytotoxicity. Mechanisms for cytoprotection.

2-Chloroacetaldehyde (CAA)-induced cytotoxicity in isolated hepatocytes was enhanced markedly if hepatocyte alcohol or aldehyde dehydrogenase was inhibited prior to CAA addition. Hepatocyte GSH depletion, ATP depletion and lipid peroxidation by CAA were also enhanced markedly. Furthermore, CAA was about 10- and 70-fold more cytotoxic than its oxidative or reductive metabolite chloroacetate or chloroethanol, respectively. Nutrients such as lactate, xylitol, sorbitol or glycerol, which increase cytosolic NADH levels, prevented CAA cytotoxicity in normal hepatocytes but further enhanced cytotoxicity toward alcohol dehydrogenase inactivated hepatocytes, suggesting that increased cytosolic NADH reduces CAA via alcohol dehydrogenase in normal hepatocytes but prevents CAA oxidation in alcohol dehydrogenase inactivated hepatocytes. However, increasing cytosolic NADH levels with ethanol or NADH-generating nutrients after CAA had been metabolized also prevented cytotoxicity and caused a partial ATP recovery, whereas oxidation of cytosolic NADH with pyruvate markedly increased cytotoxicity. This indicates that cytotoxic CAA concentrations cause oxidative stress and that ATP levels can be restored if cellular redox homeostasis is normalized with reductants. Furthermore, except for fructose, nutrients that did not increase NADH did not affect CAA-induced cytotoxicity. Fructose also caused a partial ATP recovery, and its protection was prevented by the glycolytic inhibitor fluoride. Hepatocytes isolated from fasted animals were 4- to 6-fold more susceptible to CAA-induced ATP depletion and cytotoxicity. No lipid peroxidation occurred at these lower CAA concentrations. Furthermore, all nutrients, including alanine, glutamine and glucose, prevented cytotoxicity toward hepatocytes isolated from fasted animals. The susceptibility of hepatocytes to CAA cytotoxicity, therefore, depends on both cellular redox homeostasis and cellular energy supply.

Molecular mechanisms of chloroacetaldehyde-induced cytotoxicity in isolated rat hepatocytes.

2-Chloroacetaldehyde (CAA) induced a loss in hepatocyte viability in a concentration- and time-dependent manner. Three phases before cytotoxicity ensued could be distinguished. Glutathione (GSH) was depleted immediately upon addition of CAA but only partial depletion occurred with subtoxic CAA concentrations. GSH-depleted hepatocytes were much more susceptible to CAA toxicity, indicating that CAA was detoxified by GSH. The second phase of changes involved a steady decrease in protein thiol levels, mitochondrial respiration, transmembrane potential and ATP levels. The third phase involved lipid peroxidation which commenced at around 60 min with a CAA concentration that caused 50% cytotoxicity in 120 min. Addition of antioxidants (diphenylphenylenediamine, butylated hydroxyanisole) and iron chelators (desferoxamine) at 40 min prevented lipid peroxidation and delayed CAA-induced cytotoxicity without restoring protein thiols, hepatocyte respiration or preventing further ATP depletion. Addition of dithiothreitol at 40 min, however, restored protein thiols and hepatocyte respiration, and prevented further ATP depletion and cytotoxicity. CAA-induced hepatocyte cytotoxicity therefore involved reversible thiol protein adduct formation, mitochondrial toxicity and lipid peroxidation.

Molecular mechanisms of dibromoalkane cytotoxicity in isolated rat hepatocytes.

The cytotoxicity of dibromoalkanes to isolated hepatocytes was proportional to the dibromoalkane concentration and increasing chain length of the dibromoalkane (C2-C6). The rapid hepatocyte glutathione (GSH) depletion which occurred upon addition of the dibromoalkanes was also dependent on the concentration and chain length of the dibromoalkane. When added to hepatocytes, dibromoalkanes also caused a loss in protein sulfhydryl groups. After a lag period, lipid peroxidation occurred before the onset of cytotoxicity. Antioxidants or removing the oxygen from the medium markedly delayed dibromoalkane cytotoxicity. Bromoaldehydic metabolites formed by cytochrome P450-dependent mixed-function oxidases were probably responsible for lipid peroxidation as deuterated 1,2-dibromoethane (d4-DBE) induced less lipid peroxidation and was less cytotoxic even though GSH was depleted as rapidly and as effectively. Hepatocytes were also more resistant to dibromoalkanes if cytochrome P450 isoenzymes were inactivated with SKF 525A or methyl pyrazole. Furthermore, hepatocyte susceptibility to dibromoalkanes was increased markedly if aldehyde dehydrogenase was inactivated with disulfiram, cyanamide or chloral hydrate. Cytochrome P450-induced hepatocytes isolated from pyrazole-, phenobarbital- or 3-methylcholanthrene-pretreated rats were also more susceptible to dibromoalkanes. These results suggest that dibromoalkane-induced cell lysis is due to lipid peroxidation as well as cytochrome P450-dependent formation of toxic bromoaldehydic metabolites which can bind with cellular macromolecules. Dibromoethane GSH conjugates also contribute to DBE cytotoxicity as depleting hepatocyte GSH beforehand increased hepatocyte resistance to DBE but not other dibromoalkanes.

Synthesis, cytotoxicity, hypolipidemic and anti-inflammatory activities of amine-boranes and esters of boron analogues of choline and thiocholine.

Boron analogues of carbamoylcholine and thiocholine and esters of these analogues were prepared. These compounds were fairly stable toward hydrolysis and demonstrated moderate anti-inflammatory and hypolipidemic activities in mice. The hypolipidemic activity of the compounds at a dose of 8 mg/kg/day was equivalent in reducing lipid levels in serum to those of clofibrate at 150 mg/kg/day and lovastatin at 8 mg/kg/day. The compounds demonstrated significant cytotoxic activity against the growth of murine and human tumor cells; all were active against the growth of human HeLa-S3 uterine suspended cells, and some were active against murine L1210 lymphoid leukemia, human Tmolt3 leukemia cells, colorectal adenocarcinoma, KB nasopharynx, osteosarcoma, and glioma. These studies demonstrated that antimetabolite analogues of acetylcholine exhibit the same types of pharmacological activity as other boron-substituted betaine and amino acids. Furthermore, a strong positive correlation exists between hypolipidemic activity and cytotoxicity for these new choline derivatives, as has previously been demonstrated for other boron-containing amino acids, amides, esters, and peptides.

Synthesis and antineoplastic activity of some cyano-, carboxy-, carbomethoxy-, and carbamoylborane adducts of heterocyclic amines.

Boron analogues of piperidine, piperazine, morpholine, and imidazole proved to be cytotoxic against the growth of murine and human tissue culture cells. Significant activity was demonstrated for single-cell suspensions of L1210 lymphoid leukemia, Tmolt3 lymphoblastic leukemia, and HeLa-S3 cervical carcinoma. Trimethylamine-imidazole carbonyldihydroborane 17 demonstrated activity against solid tumor growth of human colorectal adenocarcinoma, KB nasopharynx, and osteosarcoma. In addition, 4-methylpiperidine-carbomethoxyborane 12, 2-methylimidazole-3-cyanoborane 16, and 1-methylimidazole-3-(N-ethylcarbamoyl)borane 19 were active against the KB nasopharynx growth. Piperidine-cyanoborane 2, piperidine-carboxyborane 4, and 1-methylimidazole-3-(N-ethylcarbamoyl)borane 19 were effective in reducing the growth of osteosarcoma cells. The imidazole derivatives 13-19, as well as 4-methylpiperidine-carboxyborane 11 and carbomethoxyborane 12, demonstrated good activity against lung bronchogenic and glioma growth. In the in vivo studies, N-methylmorpholine-carboxyborane 7,4-phenylpiperidine-carboxyborane 9, 4-phenylpiperidine-carbomethoxyborane 10, 4-methylpiperidine-carboxyborane 11, imidazole cyanoborane 14, and 1-methylimidazole-3-carbomethoxyborane 18 demonstrated the best activity against Lewis Lung growth and P388 lymphocytic leukemia growth in mice. Mode of action studies in L1210 leukemia cells demonstrated that piperidine-carboxyborane 4 and N-methylmorpholine-carboxyborane 7 inhibited DNA synthesis, purine synthesis at PRPP amido transferase and IMP dehydrogenase sites, and thymidine kinase and thymidine diphosphate kinase activities, while lowering d(NTP) pool levels. Also, DNA strand scission was evident after incubation with these drugs.