Elsevier

Toxicology

Volume 282, Issue 3, 11 April 2011, Pages 112-121
Toxicology

Cytotoxicity and accumulation of ergot alkaloids in human primary cells

https://doi.org/10.1016/j.tox.2011.01.019Get rights and content

Abstract

Ergot alkaloids are secondary metabolites produced by fungi of the species Claviceps. Toxic effects after consumption of contaminated grains are described since mediaeval times. Of the more than 40 known ergot alkaloids six are found predominantly. These are ergotamine, ergocornine, ergocryptine, ergocristine, ergosine and ergometrine, along with their corresponding isomeric forms (-inine-forms). Toxic effects are known to be induced by an interaction of the ergot alkaloids as neurotransmitters, like dopamine or serotonin. Nevertheless data concerning cytotoxic effects are missing and therefore a screening of the six main ergot alkaloids was performed in human primary cells in order to evaluate the toxic potential.

As it is well known that ergot alkaloids isomerize easily the stability was tested in the cell medium. Based on these results factors were calculated to correct the used concentration values to the biologically active lysergic (-ine) form. These factors range from 1.4 for the most stable compound ergometrine to 5.0 for the most unstable ergot alkaloid ergocristine. With these factors, reflecting the instability, several controverse literature data concerning the toxicity could be explained.

To evaluate the cytotoxic effects of ergot alkaloids, human cells in primary culture were used. These cells remain unchanged in contrast to cell lines and the data allow a better comparison to the in vivo situation than using immortalized cell lines. To characterize the effects on primary cells, renal proximal tubule epithelial cells (RPTEC) and normal human astrocytes (NHA) were used. The parameters necrosis (LDH-release) and apoptosis (caspase-3-activation, DNA condensation and fragmentation) were distinguished. The results show that depending on the individual structure of the peptide ergot alkaloids the toxic properties change. While ergometrine as a lysergic acid amide did not show any effect, the peptide ergot alkaloids revealed a different toxic potential. Of all tested ergot alkaloids ergocristine was the most cytotoxic compound inducing apoptosis in human kidney cells starting at a concentration of 1 μM in RPTEC. Uptake studies underline the cytotoxic properties, with an accumulation of peptide ergot alkaloids and no uptake of ergometrine.

The results represent a new description of effects of ergot alkaloids regarding cytotoxicity and accumulation in human primary cells. For the first time apoptosis has been identified besides well described receptor effects. This gives a hint for a more complex mode of action of ergot alkaloids than described in literature so far.

Introduction

Since the Middle Ages ergot had a great impact in terms of mass poisoning (Schiff, 2006) or pharmaceutical approaches (Tfelt-Hansen et al., 2000). The toxic compounds, the so called ergot alkaloids, are secondary metabolites produced by the fungal family of Clavicipitaceae with most notably Claviceps purpurea parasitizing wheat, barley, and most dominantly rye. The fungus infests the living plant at the time of flowering, forming a persistent dormant body, the sclerotia. Sclerotia are resistant to temperature and remain alive during winter, or during storage of crops. In spring the sclerotia germinates and the produced spores can infect other plants again, continuing this cycle (Guggisberg, 1954).

The consumption of contaminated cereals leads to several illnesses, which result in the discovery of the relevant compounds for this effect. The sclerotia also contain the toxic metabolites of the fungus, with more than 40 different alkaloids, known so far. The intoxication was described in mediaeval literature as St. Anthony's fire, where vasoconstriction and gangrene, as well as neurotoxic symptoms were reported. Today the disease is referred to as ergotism, which can occur in two different forms, the ergotism gangrenous and the ergotism convulsive (Barger, 1931). The convulsive form causes several effects on the central nervous system resulting in spasm, hallucinations or epileptic fits. The gangrenous form (St. Anthony's fire symptoms) affects the poor blood supplied parts of the body, like fingers or toes as the main target. This results in several circulation disorders and ultimately the occurrence of necrotic, black coloured tissue and the loss of the affected body parts (Berde and Stürmer, 1978).

The relevant substances are classified by referring to their structure as clavines, lysergic acid, lysergic acid amines and ergopeptides. The clavines are also called the 8-ergolenes due to the double bond position and the other derivatives belong to the group of the 9-ergolenes. In 2005 the European Food Safety Authority (EFSA) assigned six ergot alkaloids as the most important ones (EFSA, 2005), namely ergotamine, ergocornine, α-ergocryptine, ergosine, ergocristine as peptide ergot alkaloids and ergometrine a lysergic acid amide. The structures for the individual compounds are summarized in Fig. 1.

It is well reported that the toxic effects are caused by the structure similarity of the ergot alkaloids to several neurotransmitters (Eich and Pertz, 1994, Schiff, 2006). Ergot alkaloids can act as partial agonists and antagonists of α-adrenergic, dopaminergic and serotonergic receptors and therefore have a manifold mode of action on the human body, ultimately leading to the toxic effects of the ergotism. Due to effects on the neurotransmitter receptors, the substances have also caused a strong influence on the pharmaceutical industry (Tfelt-Hansen et al., 2000). For example ergotamine with its α-adrenoreceptor-blocking mode of action, first described by Dale (1906), verified by Sachs and Yonkman (1942), is used for the acute therapy of migraine for over 50 years (Tfelt-Hansen et al., 2000).

Various animal experiments lead to several LD50 values and many epidemiological descriptions of ergotism are also available (Barger, 1931, Chaumartin, 1946, Guilhon, 1955). Different in vivo studies regarding acute toxicity have shown different sensitivities depending on the used animal species, application form and ergot alkaloid. Griffith et al. (1978) have reported rabbits to be the most sensitive species with LD50 values between 0.9 and 3.2 mg/kg b.w. after intravenous injection with ergometrine as the least toxic compound and ergocryptine as the most toxic one. These results clearly differ from other species like mice or rats with intravenous LD50 values between 30 and 275 mg/kg b.w. whereas the LD50 value for an oral dose could often not be calculated due to a very low bioavailability of the peptide ergot alkaloids (Little et al., 1982, Tfelt-Hansen et al., 2000). For humans the toxic effects are referred to the receptor interaction resulting in the acute toxic symptoms of the ergotism, such as vasoconstriction, uterus contraction, lethargy or depression or acute intoxication including diarrhea, collapse and vomiting (Guggisberg, 1954). For in vitro experiments only limited data are available for the single substances and their toxic effects on human cells. Most data consist of receptor interaction analysis for single substances in dopamine over expressing cells or tumour cells (Larson et al., 1995, Larson et al., 1999). Experiments using a neurite outgrown model by Oda et al. (2008) indicated a different toxic potential for peptide ergot alkaloids and lysergic acid amide alkaloids.

The aim of this work was to determine the toxic effect of the six mentioned ergot alkaloids using human cells in primary culture. These cells are directly isolated from the corresponding tissue and it is possible to cultivate them over a few passages. The cells remain unchanged and are therefore not immortalized or modified giving a closer look to the in vivo situation than normal cell lines. As presented in Königs et al. (2009) there was a major influence regarding trichothecene mycotoxins in comparison between cell lines and primary cells.

To study the influence of the ergot alkaloids on primary cells several parameters like cell viability, necrotic effects (LDH-release) uptake of the ergot alkaloids and triggering apoptosis (caspase-3-activation, sub G1 formation, DNA condensation) were measured. Two cell types, the renal proximal tubule epithelial cells (RPTEC) and normal human astrocytes (NHA) were chosen for a screening of the six main ergot alkaloids. The toxic relevant compounds should be identified and effects besides the well described receptor interaction were investigated. This screening gives the possibility to evaluate and compare the toxic compounds with each other. As the data for toxic effects on humans for the individual compounds are missing and the data from animal studies are difficult to compare with the human situation, the analysis of the effects on primary cells will complete the data to understand the toxicity of ergot alkaloids and their range of effects.

Section snippets

Chemicals

Ergot alkaloids ergocornine, α-ergocryptine, ergotamine-d-tartrate and ergometrine-maleate were purchased from Sigma–Aldrich (Steinheim, Germany). Ergosine and T-2 toxin were biosynthetically prepared and isolated in our lab (Beyer et al., 2009, Franzmann, 2010). Ergocristine, as well as ergocristinine, ergosinine, ergotaminine, ergometrinine, ergocorninine and ergocryptinine were purchased from Alfarma (Černošice, Czech Republic). Methysergide maleate was ordered from Biotrend (Wangen,

Stability of ergot alkaloids

Several ergot alkaloids are commercially available as pure compounds. But the characteristic chemical structure shows two different optically active carbon atoms at position C-5 and C-8 (Hafner et al., 2008). Especially the stereochemistry at position C-8 can change from the lysergic 8-(R) isomer to the so called isolysergic 8-(S) form (Komarova and Tolkachev, 2001). Chosen suffixes for these forms are -ine and -inine. Both forms can be converted into each other depending on parameters like

Stability

The experiments in cell cultivation medium clearly demonstrate that it is essential to determine the stability of the substances before performing any experiments. As presented in literature there is a known epimerization of ergot alkaloids and the formation of an equilibrium between both isomers (-ine and -inine-form) was also described (Kreilgard and Kisbye, 1974a, Kreilgard and Kisbye, 1974b, Smith and Shappell, 2002, Hafner et al., 2008). The results of our studies have shown that all the

Conflict of interest

None.

Acknowledgement

We thank the NRW Graduate School of Chemistry for financial support.

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