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    Natural Toxins Chapter 15 in Clarke 2011

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    Jocel Mae Alsola Clarin

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    Clarke’s Analysis of Drugs and Poisons in pharmaceuticals, body fluids and postmortem material FOURTH EDITION (2011) Consulting Editors Anthony C Moffat M David Osselton Brian Widdop Executive Development Editor Jo Watts Chevpter 1S: Natural Toxins iF ade. Wolff ant +A, dle Wolff Pr: Luy3 - 259 (RP) london + Chicogo Published by Pharmaceutical Press 1 Lambeth High Street, London SE1 ZIN, UK 11559 St Paul Avenue, Gurnee, 60031, USA {© Pharmaceutical Press 2011 321, 1325-1332 © TICTAC Communications (Chapter 13: Figures 13. (BP) i, ade mark of harmsceta res Pharmacare isthe publishing dvkon ofthe Royal Pharmaceta Society First edition, edited by EGC Clarke, published 1969 (Vol, 1) and 1975 (Vol. 2) Second edition (in one volume) published 1986 ‘Third edition published 2008 Fourth edition published 2011 ‘Typeset by Thomson Digital, Noida, India Printed in tay by LEGO SpA. SBN 978 085369 7114 Allright reserved. No par ofthis publication may be reproduced, stored in a retrieval sytem, ‘or transmitted in any form orby any means, without the prior written permission of the copyright rl icine meron ese wh ep i ft on oad ini tok aco sy pl esponsibility ‘errors or omissions that may be made. silent ‘catalogue record for this books availabe fom the British Library. CHAPTER: 15 Natural Toxins J F de Wolff and F A de Wolff Introduction ‘The tem ‘natural toxins usally refers to potentially toxic onganic compounds of natural origin, in contrast to mineral poisons andy thetic drugs, Sourees of thes one ange rom simple mierooganans to highly developed vertebrates. and thee chemical suture at cor reiting dri Ear tals ny ea ete tells longterm symptonss that ast almost aay organ system. The Tigh varied chemical, biological and cinial nature of tis dass of ss mean tha he conteibution ofthe anata toxiologiat to the non therapy and follow-up of ‘naturally poisoned patent limes. ‘An extensive number ofsubstaces tht comply with the dfiton of satura toxins have been used or therapetic purposes Cas examples ateergotamine, salicylic aid andthe cardia conde, sach as digoxin, dnd penn. Mote reent examples are cichosporin and borinam toxin. Most of then are not dcused inthis chapter. Silay, com aly abused sbxtances of tural origin such canna, Cosine a morphing, are covered ekewhere inthis book The dieray in chemical composition of raturaloxns prevents an arcagementacordig wo chemielsrtures. Therefore & biog Clscation hasbeen chosen Substances ate dscueed that origate from: Bacteria Fung 1 Higher plans 2 Invererates, f Nercrates Bacteria The impact of pathogenic bacteria on human health most, if not always, results from their ability to produce microbial toxins. For this chapter, a selection has been made of three common potent bacterial toxin: tetanus toxin, botulinum toxin and verotoxin. The former two are related) neurotoxic proteins produced by several Clostridium spp. Strain; verotoxin is produced by certain Echerichia col strains. ‘may also be the source of toxins previously attributed to isms, such as tetrodotoxin (TTX), which is found in pater nost probably produced by commensal microorganisms. For reasons of convention, TTX is discussed inthe section on fish poisoning. The same holds for those freshwater cyanobacteria that produce saxi- toxins, which are described in the section on mollusc poisoning, Clostridia (botulinum and tetanus) Botulinum and tetanus neurotoxins are produced by strictly anaerobic Sacer loging the gn Cla and case the neropaac syndromes of botulism and tetanus. The botulinum toxins consist of at least four peptides with molecular sizes that range from 150kDa to, SOLD The tea tox has two dlink peptide chains of toler sie SDs and 100KDa, The costal neurotoxins are the Test potent oun koow with an etinted median lethal dose of ‘bout ng/g intrvcrnady,Botliauns ons are fod poison, whereas the ca xin no, Teams flows the entiation of nec ‘wun wih spores of Ct tet Tetanus sae in couse with immunisation programmes, but it has a high ease fatality ate of| 24%, An estimated 00000 newinorns de from neonatal tetanus world wide ach yea. Treatment with antitoxinand intravenous administration ‘of peniilin soon after infection may reduce mortality. Generally apli- ‘able analytical methods for the ientifation and quantification of tetanus toxin have not been reported (Che ‘Clase food-borne botulism occur afer ingestion of food contam- inated by prelormed toxin of C: botulinum. The cinical presentations are stereotypical. Within 12-36h of ingestion, the patient develops ‘iplopia and ptosis, followed by a descending pattern of weakness that affects the upper and then the lower limbs, and respiratory paralysis in Severe cates, There is noapic treatment for botuliss recovery isnot uncommon bat it requires the regeneration of new motor endplates, which takes weeks. Food:-borne botulism remains a matter of concern, EXfetive treatment depends on provision of intensive cate and rapid administation of botulinum antitoxin based on clinical presentation, and should not wait he results of time-consuming laboratory diagno sis (Sobel 2008). Owing to its extreme neurotoxicity and easy produc tion, botulinum toxin has also Been suggested asa posible agent In bioterrorism (Villar etal 2006) Laboratory proof of botulism requires the detection ofthe toxin or bacterial deoxyribonucleic acid (DNA) in the patient's blood or stools. Its sl available, the suspected food should ao be tested for the toxin. With an old toxin identiieation method using a mouse toxicity test with anttoxin neutralisation, laboratory results are not always confirmatory, A numberof immunoassay methods have been reported for the detection of botulinum toxins. A radioimmunoassay has been replaced by a more sensitive ‘double sandwich” entyme- linked immunosorbent assay (ELISA) method based on application ofthe antibodies. An ELISA amplification technique may generate a To-fold increase in sensitivity, rendering it suitable for diagnostic purposes (Hatheway and Ferteira 1996). A promising new method for the detection of bacterial spore and toxins uses a biosensor based fon electrochemiluminescence. Toxins and bacterial spores are cap tured on antibody-conjugated micromete-sized magnetic beads, (0 lowed by binding of uthenium(l}-trbipyeidal chelate-labeled reporter antibodies. Immunomagnetically captuced target material ae then collected on a magnet. Electrachemiluminescence is evoked from the Ru-taged reporter antibodies by application ofan electric potential. Using a commercially available analyset (ORIGER), a fem togram sensitivity levels ebtaned for a numberof toxins, incluin botulinum toxin. For Bacillus anthracis, this epcthod yields a limit ‘of detection of 100 spores (Gatto-Menking etal. 1995). Even more Promising are laboratory methods based on the measurement of clostridium DNA by polymerase chain reaction (PCR) in combina tion wit capillary or gel electrophoresis or other detection methods Combined with ELISA or probe hybridisation, detection limits a low 380.1 spore per gram of feces or food have been reported (Lindstrdm and Korkeala 2006). Escherichia coli Several strains of the common intestinal bacterium E.coli may cause diarrhoeal disease in humans. One such strain, which occurs naturallyin the gut of cattle and other animals, produces a potent cytotoxin: vero- toxinor verocytotoxin, This E.coli strain, usually referred toas VTEC. is 243 244 Natural Toxins ‘geographically widespread and is associated with life-threatenit hhuman diseases that range from bloody diarthoea to the haemolytic uraemic syndrome and thrombocytopenic purpura. Young, children, ‘pecially those from urban areas, area particularly vulnerable group because of their age and immunological naivety to farmhouse infections (Chattopadhyay etal. 2001; Parma etal. 2000; Pritchard era 2000) ‘The chemical nature of verotoxin has not yet been elucidated. Verotoxin-producing E.coli strains are characterised through PCR and DNA hybridisation, and by a Veto-cll cytotoxicity assy (Leung, et al, 2001), Other E: col strains (015747) and other bacterial species produce Shiga toxins, which may produce disease in humans after consumption of undercooked contaminated beet. In humans thee toxins may cause hhaemorthagic colitis and haemolytic uracmic syndrome. Enzyme {immunoassays ae available that detect Shiga toxins in diarthoeal stool samples from humans and in contaminated beef (Atala et al. 2000; Hyatt et a, 2001). Fungi Mycotoxins are a group of potentially toxic substances produced by species of the botanical class Fungi. Remarkably, only avery few mush- room species are deadly ater ingestion, Many more fungi that grow on food may produce toxins that do not, generally speaking, cause symp- toms of acute intoxication, but that may cause adverse effects after chronic ingestion of small quantities. In this section, selection is made of fungal toxins from Aspergillus, Fusariwn (Gibberella zeae) and CClaviceps genera that have a possible impact on human health, where the analytical toxicologist may have an important role in the prevention of food-borne diseases. In addition, the toxic principles from some of ‘the most common toxic mushroom species are described Aspergillus Aflatoxin Allatoxin (Fig 15.1) is a family of mycotoxins, produced by the mould “Aspergillus flavus, that are found as contaminants in both human food- stuffs and animal feed, particularly in maize, groundnuts and other nuts such as pistachios. The aflatoxin-producing Aspergillus species are ubiq- uitous in areas with a hot, humid climate, Foodstuffs may be contam- inated with aflatoxin both pre- and post-harvest. Exposure occurs ‘through consumption of contaminated food and also during the hand ling and processing of aflatoxin-contaminated crops, Aflatoxins have been detected in human milk, urine and blood samples (IARC 19933). Figure 15.1. Aflatoxin-My ‘Aflatoxin-B, isa very potent human carcinogen that reacts with DNA ‘once it has been bioactivated to the epoxide. It is thought to cause hepatocellular carcinoma. "Aflatoxins have been measured in human blood by high performance liquid chromatography (HPLC) with fluorescence detection (Abdulrarzaq et al. 2002). In 201 samples of umbilical cord blood, aflatoxins were detected in 110 and there was a significant negative Correlation between birth weight and the aflatoxin concentrations found. Kussak etal. (1995) applied a liquid chromatography-tandem mass spectrometry (LC-MS{-MS}) system to measure aflatoxins in spiked urine samples and achieved detection limits of 2-10pg. Anum. ber of analytical methods have been described for the analysis of aa. toxins Gy, Gz, By and By in foodstus. High performance thin-layer chromatography (HPTLC) and ELISA procedures have been described, x swith fluorescence detection is considered tobe the a ee 1998; Gilbert 1993; Holcomb et al 1997, ay sttoty choi din Jaimer et a. 2999)" st these methods is presente al. (2009, 8-4 review of the Textaction depend greatly on the physics ere ined mati atOtn esolble ggg ree miture of acetone, chloroform or metho . vealed for tetatin. Farther parten, se al by solid-phase extraction followed by conca Be accompli epic anal. Reversed-phase HPLC with ms Sherer tonne 8nd seerton oft yar eantageof these aqueous media is that the natural oo aie aleh hem so readily detectable by TLC, is quenched Th wn eam by post-column derivatisation with iodine, «py Fully adopted 26 an oficial method by the Association of an ct anites (AOAC) and the International Union of Pure anda Chemisty (IUPAC) in 1981 (AOAC 1991). Although much meet frown about afatons ochratoxin is gradually being recogni ct anya toxin of equivalent importance (Bayman and Baker 3006) Ochratoxin ‘Ochratoxin-A (OTA, Fig. 15.2) is a widespread mycotoxin produc rainly by the fungi A. ochraceus and Penicillium verruculosum du the storage of cereals, cereal products, herbs, spices (LAR 1993; Petzinger and Ziegler 2000) and other plant-derived produc such a coffee (Viani 1996). OTA is chlorinated, which is unusual fra natural product. Ochratoxin-B, which is not chlorinated, and oc. toxin-C, the ethyl ester of OTA, are ess toxic. OTA has been reported bbe nephrotoxic, immunosuppressive, carcinogenic and teratogenic ia animal studies. However, a with aflatoxins, it soften not lear towha ‘extent animal studies are applicable to toxicity in humans. r 1 4 solvents, sul polar solvents can = tony tao thane OH oO “CHy a Figure 15.2 Ochratorin-A, Ochratoxin-A has been found primarily in northern temperate bi ley- and sheat-growing areas, but the presence of OTA in grea processed coffee has received much attention recently. As the lit OTA-producing fungi has expanded, so has the list of foods that >be contaminated with ochratoxins (Bayman and Baker 2006). reagsumption by pigs of mouldy pig feed may result in deste levels of OTA in pork-derived products. Since OTA is hydrolysed by ricinal Aloray itis unlikely to be found in milk or meat from ot (Seu oe When ingested by humans, OTA i pea in ¢limination half-life of about 35 days attributed to binding to pls potins Daring the 1950s a tal chon Tay i it several counts inthe Balkans and became Ja acdemic nephropathy (BEN). BEN has a striking si 0 Pepa RPbropathy induced by OTA. The causative factors boos, Teeth OTA levels in the diet. The torn is also 0 possible arc Weer anda immunotoxic agent. tappears to exertits toxic Premotng an increased level of lipid peroxidation, by inhibition lation rection and possibly by conversion int© ‘that are capable of bin or the any ing DNA (Marquardt et a. 1990) and Dick [re &f OTA in blood, serum and milk samples Zi, freee 93) 4 immunoaffinity column clean-up and weet 100) aspen a Ceo ban» ito Sgt ie ‘The Oe and limitof deren CYHPLC with fluorescence detection. Seer gto (LOD) values were 005 and O01 BEE tion and meee i Procedures have been proposed forthe we ssutement of OTA in food products as vie" ta ase HG wi hres aun Cp Beaten Nee een oe atgmandn min ware ea hed nce be Ge ge eh aot Seen his he ar bess a mean es ein Fusarium Deorynivaleno! Devtrialenol (DON) isa mycotoxin belonging tothe teichothscene typeaoxins produced by Fusurium gamancuur (Caerlrsom sod Fadmorum. These ung’ grow om ceeacops mained aoe {UARC 199%). Aswheat and maize products orm comidesble partof the viet in many repons, the toucity and the content of DOW sre important fses in food safety conta eoxpivalenal cin affect the immune system and both suppreson an activation have ben reported, but the toxin snot considered te mutagenic or carcinogen “wo outbreaks of human disease related to trchothecenes have been report, one in China in 1984 to 1985 and one in nia in 1987, Cnsurption of mouldy wheat or maize reste within 3-30 min in Sse, vomiting. abdominal pain lrrhoen,direness and headache, DON has been tected inthe cea in a range of 034-923 mh together with 0001-0387 mg/kg zearalnone (IARC 19930) (oe ates), ‘Tetolerabl daly intake (TD) n humans considered tobe 0.5 Ug tery mas per day {Health Council of The Netherlands 2001), An erie of sme ofthe major mycotoxin, Including DON, has been fivenby Richard (2007), lack eal (1986) developed a gas chromatography-massspctro- metry (GC-MS) procedure forthe measurement of trace quanies of trichotecene mycotoxin in sample af Blood and urine. The method involved purifiestion by reversed-phase Sep-Pak Cy cartidges and drratsation tothe heptaflorobutyl esters LODS fora range of mycotoxins ranged from 2 ppb to 7 pp My etal (2003) explored tinnary measurement of BON as 4 potenti biomarker of human tapos. Urine samples were treted with Bglucuronidase and sul- {ntsc enrymes to release conjugated DON, which was then analysed byan immtunnafinty-calumn-HPLC technique Positive esl we Sted by 151, Ure samples eal rom eras vig S potently high-exposure region of China and a low-exposure raion mee compared. Mean levels in samples from the suspected Iigh- and low-exposue regions were 37 ng range 14-94 nga) and ng/ml (eange 4-14 glad, respectively ‘A method forthe quantification of DOW in whest using immano- sn una an HPLC wth evil (OY detection as been pushed (Cahill eal 1999). Te ompares very well with he efston capture detection (ECD) method publ by Tske and per (1985) Nowadays, HPLC with UY detection i the recom ime method for tesde analasinfood (Straka et a, 2008) However move sophisticated method combining LCS with atm spheric pesurephetoiontsation hasbeen recently described by Tanaka {ta (209) forthe detection of roel and DON in whet. Th ‘edu the mart eects sem with ther methods and ave LODSof0.2 nd 039 ng fr nivaenol and DON respectively 12 toxin Awe rl mean the wichotecens cogent 12 ox Droduced by F. spovorchiades.T-2 toxin has Been suggested a5 2 Potena chemical warfare agent (Hodgson «tal. 1998). A disease oven as alimentary toa aeukia fe associated seth T2 toxin, Ih severe outbreaks of poisoning ths disease has. fatality rate as high as Aleukis i charaterned by hyperacmia of the oral mucosa, wetknes fever masses and vomiting, and i severe cases by Fungi 245 sastroenterts In ater stages ofthe disease leukopenia, granulopenia nd progressive Iymphocytoss occu, followed by severe hacorrha- gic diathess, necrotic pharyngitis and laryngitis, and anemia. In an ‘outbreak in Kashmir i 1987, T-2 toxin concentrations in flour sam= ples were found to be 0.55-0.8 mg/kg (IARC 198d}. The analytical Aspects of T-2 toxin are discussed in the next section, which describes {general methods for the analysis of trichothecenes Zearalenont 2Zearlerone isa benroxayeloetraecin derivative produced in F. gr imine (Cilla se) an reated species, and primal ah ited with maize (Zo mast sarong the most widely dsibted tmyotonins. Toi ees in humane ar exec dificult to ames Jnvasr of contaminated cre evr mycotoxins ae presets ttmcmaly Epon dasuphng eset ocarinaninal Gu these wet nat eeported inthe two ores devtiba shove, n which bth DON nd reartenone were Blenone toxic ws Zomdeiang ateedtolis W sav, cnat tert watregee sth promoter that ned for ie info pucing ania in E2'Enropean Union. Gonseuerys methods have been devised for ‘kctrmination ofthe patent compound snd ts metabolite io bovine brine Schmid eal (208) applied LC-MS(CMS) to screen for stones nd seonal and eartenone in utine, and were able to detect and “ant tho tubstances at eels below Lgl ‘A humans ae sual expose tos name of fisriam toxins through contaminated cereals mst atl methods forse on these foodstts have been developed to idestfy and quantify 4 amber of rnycotonins in asingle procedure, Theappiaion of Tin myeoroxn mals hen reviewed (Lin a. 1988). TU Screning of mycotoxins in fod wth detection for aflatoxin in ee 03 gfe ochrstosn-A im pork Kidney and 20g serlenone i erat Most quantitative methods ave been tose on GC-ECD, but HPLC hoe allo heen applied. Keka (1398) dese lensp proce for the aals f esalenoe naire Sndtrichthecenes mest he microgram per lgeam rang, based ‘and mulifntiona dean-ap comms. A method ative and. quantitative GC'MS sna of eiht Aifeent myextoinincadg DON, F-2 toxin and tearlenone as described by Oni era (1998), th mie of detection of 01-03 Snd recoveries of around 90%. Anaya methods forthe foam toxins DON and 1-2 toxin wee reviewed by Koch 200). With the onsiderableavansesin AS ecsogy sce tat ime theaters iecoming the major technique fr multinals of myotenins in food sal For example, Sly oa (200) were able wo develop a 16 MS (EMS) method forthe determination of 39 mycotoxin in wheat and Imsine,inliding rearienone and rested deraties Because ofthe ‘henicl diversity ofthe anayten, uth postive: and neptve ton ees {rosea ionisation (SD modes were api in wo comectiv hro- imatogripic runs. It was possible by enracting samples with mtore acon, water and ssi ach (792201) to inet ta extracts GilstedI+1 without forther ceon-op. More tceny Diana Di Maveng ct (2009) reported onthe tse of LCATSUNTS) with pos tiveion EI fr the mulanalwso mycotoxin in fost such ah soy isoflavones, St ohn’ wort gai, Ginko Bl a Sack rash They found the optimam eration soem to bea mitre fen acetate and formic sil (95:5) and puri the extract by a combination of iguidcguid parton with hexane and solidphase extraction car ses LOps "and 10 wt nthe ngs 030 an Tog respectively. Claviceps purpurea Poisoning by ergot allaloids produced bythe mould Clavces purpurea is usualy reed to as eyorom, and i probably the obit seconded food-borne dscse of fungal origin Cl. purpurea grows on food grains parcully rye, during wet seasons, Srmptoms of ergotism include {sythems, Garocs, vomiting « burning sensation ofthe lbs and, tvenual, gangrene. Centel nervous system’ (CNS) effets are 246 Natural Toxins convulsions, catalepsy, dullness or maniacal exeitement. These symptoms ‘canbe explained by the pharmacological properties ofthe engot alkaloids, nag ‘which may cause an cadrenengic blockade, as well as serotonin oonism. The ergot alkaloiels ergotamine (the best known, ergometrine, or engonoving, have been used for centuries agents to stimulate uterine contractions and in the treatment of migraine attacks. Overdose with these ages, an! intoxication with ergot alkaloids from other sources, can be treated symptomatically with potent vasodi- lator drugs, such as sodium nitroprusside, andl by maintaining adequate circulation, HG of 8 08 ON A H " So ae Co oO Hs HN Figure 15.3. Exgotamine, ‘The method of choice for analysis of ergot alkaloids in serum is HPLC with fluorescence detection after liquid-solid extraction (Moubarak etal, 1996). The structural chemistry and a comparison of the available analytical techniques for ergot alkaloids havebeen reviewed ‘extensively by Flieger etal. (1997). Kerska et al. (2008) published a review on the analysis of ergot alka- loids in cereal samples by means of LC with diode array detection and LC-MS(-MS). Mushrooms. Amanita muscaria ‘A number of basidiomycetes contain hallucinogenic principles the best- known examples being the Amanita muscaria and the Pslocybe species types. The fly agaric (Am. muscaria) is the European archetypical ‘mother of all mushrooms’ in legends and fairy tales, with a red hood and white spots Is indigenous tothe northern hemisphere, but i also found in some parts of South Africa, South America, Australia and New Zealand, Despite popular belief, no fatalities have been reported in hhumans. The most important toxin in Am. muscaria is not muscatine (which does occur in trace amounts) but ibotenic acid and its decar- Boxylation product muscimol. Poisoning with Am. muscaria usually results from deliberate ingestion to obtain a psychoactive response and symptoms occur within 20-180min. Muscimol (Fig. 15) is a "Y-aminolbutyricacid (GABA) receptor agonist tcauses CNS depression that results in drowsiness and dizziness, followed by lation, increased ‘motor activity, tremor, agitation and hallucinations, There are no spe- cific antidotes and recovery is complete upon awakening, Figure 15.4 Muscimol ‘There is no validated procedure available for analysis of ibotenic acid and muscimol in blood or urine. For their determination in mush- rooms, an ion-pait HPLC method with UV detection after methanol ‘extraction has been described (Tsunoda et al. 1993), More recently, ‘methods have been published for the analysis of ibotenic acid in dried mushrooms using GC-MS (Tsujikawa et al, 2006) and LC-MS, (Toujikawa etal. 2007). Psilocybe type ‘The ‘palocybe syndrome’ is caused by consumption of one ofthe mat species of the cosmopolitan genera Psilocybe, Panacolus, Copelendia, GymnopisorStrophara. These mushrooms contain the indole gy 4 wren psilocybin (Fig. 15.5) and its dephosph at ee wich rau ie tins 8 poten or entay congener psiloc We about 100 times less potent than Ise =

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