Aqel W. Abu-Qare

Biomarkers of apoptosis: release of cytochrome c, activation of caspase-3, induction of 8-hydroxy-2'-deoxyguanosine, increased 3-nitrotyrosine, and alteration of p53 gene.

Biomarkers rely on biochemical, histological, morphological, and physiological changes in whole organisms. Their use is becoming an important tool to examine changes at cellular and molecular levels, especially in nucleic acids and proteins. Biomarkers are used to measure exposure to a toxic agent, to detect severity of any toxic response, and to predict the possible outcome. Information on the mechanisms of action of toxicants can allow the development of potential biomarkers of effect and thus improvement of the risk assessment processes. Use of biomarkers as a tool to predict induction of apoptosis allows identification of biological signs that may indicate increased risk for disease. In cells undergoing apoptosis, the release of cytochrome c from the mitochondria to the cytoplasm and the activation of caspase-3, a key enzyme to execution stage of apoptotic pathway, have been studied as biomarkers of cell death (apoptosis). Products of DNA fragmentation that either accumulate in the cellular tissues or are excreted in the urine are useful markers of DNA damage. The induction level of urinary or cellular level of 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine has been used as a marker to measure extent of DNA oxidative damage. Furthermore, alteration or overexpression of the p53 gene was considered an indication of apoptosis. This article reviews some of the aspects of biomarkers of apoptosis, indicating relevance of their uses to predict apoptosis following exposure to environmental toxicants.

Methyl Parathion: A Review of Health Effects

Methyl parathion is an organophosphorus (OP) insecticide with insecticidal properties derived from acetylcholinesterase (AChE) inhibition; this same property is also the root of its toxicity in humans. Poisoning with methyl parathion leads to cholinergic overstimulation with signs of toxicity including sweating, dizziness, vomiting, diarrhea, convulsions, cardiac arrest, respiratory arrest, and, in extreme cases, death. Reports of methyl parathion intoxication, usually seen only in field pesticide applicators, have increased throughout the United States as a result of unauthorized application of methyl parathion inside homes. The health concerns of the use of methyl parathion have resulted in cancellation of its use in most food crops in the United States. This review examines the well-documented neurotoxicity of methyl parathion as well as effects on other organ systems.

A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine

A method was developed for the separation and quantification of the anti-nerve agent pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid, and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1-H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C(18) Sep-Pak(R) cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C18 column, and UV detection at 280 nm. The compounds were separated using gradient of 1-85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min. The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9+/-9.5, 73.7+/-9.8, 88.6+/-9.3, 83.9+/-7.8, and from urine 69.1+/-8.5, 74.5+/-8.7, 85.9+/-9.8, 83.2+/-9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats.

A Role for P-Glycoprotein in Environmental Toxicology

P-Glycoprotein (P-gp) is a transmembrane protein, playing significant roles in the process of drug discovery and development and in pest resistance to pesticides. P-gp affects absorption, disposition, and elimination of different compounds and is mainly expressed in intestines, liver, kidneys, heart, colon, and placenta. The expression of P-gp in the blood-brain barrier (BBB) has been associated with the restricted access of many compounds to the central nervous system. Generated knockout mice by disruption of mdr 1a gene, encoding for P-gp, showed that this protein was expressed in the BBB. The absence or the low levels of P-gp elevated drug concentrations in tissues and decreased drug elimination. P-gp is responsible for resistance of cells to agents, particularly the anticancer drugs, by removing these drugs from cells. Increased expression of P-gp is implicated in decreased HIV drug availability at certain intracellular sites. The role of P-gp in affecting efficacy and toxicity of environmental toxicants such as pesticides and heavy metals has not been adequately investigated. Studies showed that P-gp contributes to resistance to pesticides in certain pest species, and to decrease toxicity by removing compounds from cells in mammals. Placental drug-transporting P-gp plays a significant role in limiting the transport of toxicants such as potential teratogens to the fetus. Several in vitro or in vivo assays, including using P-gp knockout or naturally deficient mice, were described for testing P-gp modulators. The role of P-gp following concurrent exposure to more multiple compounds needs further research. P-gp modulators should be carefully used, since some modulators that reverse P-gp efflux action in vitro may lead to alterations of tissue function and increase toxicity of xenobiotics in normal tissues. Recent reports from the pharmaceutical studies on the significance of P-gp as transporters in altering the efficacy and toxicity clearly highlight the need for further research in interaction with environmental toxicants.

Sarin: health effects, metabolism, and methods of analysis.

Sarin (O-isopropylmethylphosphonofluoridate) is a highly toxic nerve agent produced for chemical warfare. Sarin is an extremely potent acetylcholinesterase (AchE) inhibitor with high specificity and affinity for the enzyme. Death by sarin is due to anoxia resulting from airway obstruction, weakness of the muscles of respiration, convulsions and respiratory failure. The main clinical symptoms of acute toxicity of sarin are seizures, tremors and hypothermia. Exposure to sarin during incidents in Japan in 1994, 1995 and 1998, and possible exposure to low levels of sarin during the Gulf War, resulted in the deaths and injury of many people in Japan and caused possible long-term health effects on Gulf War veterans. Symptoms related to sarin poisoning in Japan still exist 1-3 years after the incident and include fatigue, asthenia, shoulder stiffness and blurred vision. Sarin produced seizures in rats and pigs. Recent studies showed that long-term exposure to low levels of sarin caused neurophysiological and behavioral alterations. Toxicity from sarin significantly increased following concurrent exposure to other chemicals such as pyridostigmine bromide. Further research to examine effects of sarin on the cellular and the molecular levels, gene transcription, endocrine system as well as its long-term impact is needed.

Induction of urinary excretion of 3-nitrotyrosine, a marker of oxidative stress, following administration of pyridostigmine bromide, DEET (N,N-diethyl-m-toluamide) and permethrin, alone and in combination in rats

In this study, we determined levels of 3-nitrotyrosine in rat urine following administration of a single oral dose of 13 mg/kg pyridostigmine bromide (PB) (3-dimethylaminocarbonyloxy-N-methylpyridinum bromide), a single dermal dose of 400 mg/kg N,N-diethyl-m-toluamide (DEET) and a single dermal dose of 1.3 mg/kg permethrin, alone and in combination. Urine samples were collected from five treated and five control rats at 4, 8, 16, 24, 48, and 72 h following dosing. Solid-phase extraction coupled with high-performance liquid chromatography with ultraviolet detection at 274 nm was used for the determination of tyrosine and 3-nitrotyrosine. A single oral dose of PB and a single dermal dose of DEET or their combination significantly (P<0.05) increased levels of 3-nitrotyrosine starting 24 h after dosing compared with control urine samples. The maximum increase of 3-nitroytyrosine was detected 48 h after combined administration of PB and DEET. The ratio of 3-nitrotyrosine to tyrosine in urine excreted 48 h after dosing was 0.19+/-0.04, 0.20+/-0.05, 0.28+/-0.03, 0.32+/-0.04, 0.19+/-0.05, 0.42+/-0.04, 0.27+/-0.03, 0.36+/-0.04, and 0.48+/-0.04 following administration of water, ethanol, PB, DEET, permethrin, PB+DEET, PB+permethrin, DEET+permethrin, and PB+DEET+permethrin, respectively. The results indicate that an oral dose of PB and a dermal administration of DEET, alone and in combination, could generate free radical species, and thus increase levels of 3-nitrotyrosine in rat urine. Induction of 3-nitrotyrosine, a marker of oxidative stress, following exposure to these compounds could be significant in understanding the proposed enhanced toxicity following combined exposure to these compounds.

Inhibition of cholinesterase enzymes following a single dermal dose of chlorpyrifos and methyl parathion, alone and in combination, in pregnant rats.

Pregnant Sprague-Dawley rats (14-18 d of gestation) were treated with either a single dermal subclinical dose of 30 mg/kg (15% of dermal LD50) chlorpyrifos (O,O-diethyl O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate) or a single dermal subclinical dose of 10 mg/kg (15% of dermal LD50) methyl parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate) or the two in combination. Chlorpyrifos inhibited maternal and fetal brain acetylcholinesterase (AChE) activity within 24 h of dosing, (48% and 67% of control activity, respectively). Following application of methyl parathion, peak inhibition of maternal and fetal brain AChE activity occurred at 48 h and 24 h after dosing (17% and 48% of control activity, respectively). A combination of chlorpyrifos and methyl parathion produced peak inhibition of maternal and fetal brain AChE activity at 24 h postdosing (35% and 73% of control activity, respectively). Maternal and fetal brain AChE activity recovered to various degrees of percentage of control 96 h after dosing. Application of methyl parathion or chlorpyrifos alone or in combination significantly inhibited maternal plasma butyrylcholinesterase (BuChE) activity. No significant inhibition of fetal plasma BuChE activity was detected. Peak inhibition of maternal liver BuChE occurred 24 h after application of methyl parathion or chlorpyrifos alone or in combination (64%, 80%, and 61% of control activity, respectively). Significant inhibition of placental AChE occurred within 24 h after application of methyl parathion or chlorpyrifos alone or in combination. The results suggest that methyl parathion and chlorpyrifos, alone or in combination, were rapidly distributed in maternal and fetal tissues, resulting in rapid inhibition of cholinesterase enzyme activities. The lower inhibitory effect of the combination could be due to competition between chlorpyrifos and methyl parathion for cytochrome P-450 enzymes, resulting in inhibition of the formation of the potent cholinesterase inhibitor oxon forms. The faster recovery of fetal plasma BuChE is attributed to the de novo synthesis of cholinesterase by fetal tissues compared to maternal tissues.

Development of a high-performance liquid chromatographic method for the quantification of chlorpyrifos, pyridostigmine bromide, N, N-diethyl-m-toluamide and their metabolites in rat plasma and urine

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C18 column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1-85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2+/-7.9, 74.9+/-8.5, 81.7+/-6.9, 73.1+/-7.8, 74.3+/-8.3, 80.8+/-6.6, 81.6+/-7.3 and 81.4+/-6.5, and from urine 79.4+/-6.9, 77.8+/-8.4, 83.3+/-6.6, 72.8+/-9.0, 76.3+/-7.7, 83.4+/-7.9, 81.6+/-7.9 and 81.8+/-6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.

Effects of sub-chronic in vivo chlorpyrifos exposure on muscarinic receptors and adenylate cyclase of rat striatum

Abstract. In this study dosing regimens were designed such that cholinesterase inhibition following exposure to chlorpyrifos was produced in one treatment group, but was absent in the other. The higher dosing regimen inhibited plasma and brain cholinesterase activities by 51 and 70%, respectively, and resulted in decreased [3H]cis-methyldioxolane ([3H]CD) binding, which was attributable to a decrease in Bmax. No concomitant loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding sites was observed, indicating that the M2 muscarinic receptor subtype to which [3H]CD binds is particularly susceptible to alterations induced by chlorpyrifos treatment. As the M2 receptor subtype is surmised to be the muscarinic autoreceptor, decreases in this receptor may exacerbate poisoning by organophosphorus agents as a result of decreased ability to terminate synaptic acetylcholine release. The ability of carbachol to inhibit striatal adenylate cyclase, which is an effector molecule associated with the M2 receptor, was unaltered in chlorpyrifos-treated rats. Decreases in M2 receptors occurred with the higher dosing regimen, in the absence of any clinical manifestations. Thus, in the absence of overt clinical signs, perturbations of the muscarinic receptor system did occur as a result of sub-chronic chlorpyrifos exposure. Such alterations may contribute to neurological impairments that develop following chronic organophosphorus exposure.

Simultaneous determination of malathion, permethrin, DEET (N,N-diethyl-m-toluamide), and their metabolites in rat plasma and urine using high performance liquid chromatography

A method was developed for the separation and quantification of the insecticide malathion (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorodithioate), its metabolite malaoxon (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorothioate), the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), two of its metabolites m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, the insect repellent N,N-diethyl-m-toluamide (DEET), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method used high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 210 nm. The compounds were separated using gradient of 45--99% acetonitrile in water (pH 3.5) at a flow rate ranging between 0.5 and 2 ml/min in a period of 15 min. The retention times ranged from 7.4 to 12.3 min. The limits of detection ranged between 20 and 100 ng/ml, while limits of quantitation were 50-150 ng/ml. Average percentage recovery of five spiked plasma samples were 80.1+/-4.2, 75.2+/-4.6, 84.5+/-4.0, 84.3+/-3.4, 82.8+/-3.9, 83.9+/-5.5, 82.2+/-6.0, 83.1+/-4.3, and from urine 78.8+/-3.9, 76.4+/-4.9, 82.3+/-4.5, 82.5+/-3.9, 81.4+/-4.0, 83.9+/-4.3, 81.5+/-5.0, and 84.5+/-3.8 for, malathion, malaoxon, DEET, m-toluamide, m-toluic acid, permethrin, m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid, respectively. The method was reproducible and linear over range between 100 and 1000 ng/ml. This method was applied to analyze the above chemicals and metabolites following combined dermal administration in rats.