Elsevier

Toxicology Letters

Volume 125, Issues 1–3, 30 November 2001, Pages 125-132
Toxicology Letters

Cytotoxicity of natural compounds in hepatocyte cell culture models: The case of quaternary benzo[c]phenanthridine alkaloids

https://doi.org/10.1016/S0378-4274(01)00430-1Get rights and content

Abstract

The quaternary benzo[c]phenanthridine alkaloids (QBA) produce a plethora of species- and tissue-specific effects but the molecular basis of their biological activities remain mysterious. The objective of the present study was to investigate the cytotoxicity of QBA alkaloids, sanguinarine (SA), chelerythrine (CHE), fagaronine (FA), and the extract from Macleaya cordata in primary cultures of human and porcine hepatocytes. The cellular damage was assessed by the MTT assay, lactate dehydrogenase (LDH) leakage and the determination of intracellular glutathione (GSH) levels. The results are summarised as follows: (i) The alkaloids tested in doses 0.1 and 10 μM did not display statistically significant cytotoxicity for 0–3 h incubation; (ii) SA and CHE showed the dose- and time-dependent toxicity within the range 25–100 μM whereas FA was not toxic; (iii) the LDH leakage into the medium was higher for SA than for CHE, thus revealing a potent potential of SA to disturb cell-membrane integrity; (iv) after 3 h incubation with 100 μM SA/CHE, mitochondrial dehydrogenase activity (MTT assay) and the cellular GSH levels decreased to residual values of about 40% suggesting that mitochondria are unlikely to be a primary target for SA/CHE in the cell; (v) no differences were found in the response to QBA application in human vs porcine hepatocyte.

Introduction

Sanguinarine (SA), chelerythrine (CHE) and fagaronine (FA), are quaternary benzo[c]phenanthridine alkaloids (QBA) and display a wide spectrum of non-specific biological activities (Walterová et al., 1995). In contrast to SA and CHE, FA has a different substitution pattern in the A-ring (Fig. 1). QBA are found in plants of families Caprifoliaceae, Fumariaceae, Meliaceae, Papaveraceae, and Rutaceae (Šimánek, 1985). QBA are the elicitor-inducible secondary metabolites and are considered phytoalexines because of their antimicrobial activity.

Two standardised QBA extracts are available on the market. Sanguiritrine, a QBA extract from Macleaya species (mixture of SA, CHE and three minor QBA), serves as a remedy for internal uses in myopathy therapy and as antimicrobial drug in Russia, and sanguinaria, a QBA extract from Sanguinaria canadensis (mixture of SA, CHE and four minor QBA), is used as the antiplaque component in dentifrices and oral rinses manufactured in Europe and USA (Tenenbaum et al., 1999). Sanguinaria was used in the USA as expectorans but FDA classified this remedy as unsafe for uses in drugs (MARTINDALE The Extra Pharmacopeia, 1996). Interestingly, the veterinary preparation Sangrovit®, which contains approximately 4% of the QBA extract—sanguinaria, is advertised as a weight gain stimulant in farm animals (ANONYMOUS, 2000). FA possesses antileukemic activity (Suffness and Cordell, 1985), inhibits HIV-1 and HIV-2-reverse transcriptase (Tan et al., 1991, Kerry et al., 1998), and DNA topoisomerases I and II (Wang et al., 1993). FA is being considered as a potential antitumor drug (Kerry et al., 1998).

As with many natural compounds, QBA exhibit toxic side effects in addition to their desired pharmacological activity. Contamination of edible oils with the oil from seeds of Argemone mexicana and A. ochroleuca was suggested to be the cause for the disease epidemic dropsy (Das and Khanna, 1997). The principal constituent responsible for argemone oil toxicity was identified as sanguinarine. Damm et al. (1999) reported that the long term use of oral products containing sanguinaria appears to be associated with an increased prevalence of leukoplakia of the maxillary vestibule. However, this conclusion has been criticised (Munro et al., 1999). From in vivo experiments with rats and guinea pigs, it was concluded that the lipoid structures in the gastrointestinal tract, liver and kidneys are the target sites for SA (Tandon et al., 1992). SA and CHE adverse effects may be due to their non-selectivity towards specific target structure or to toxicity that results from metabolic activation to reactive intermediates, which are capable of covalent binding to cellular macromolecules. Their covalent binding to proteins can cause cell death by disturbing essential biochemical processes or immunological reactions, whereas binding to DNA may initiate the process leading to cancer. The positive charge on nitrogen atom facilitates the interaction binding with DNA. In this respect, it is FA that is most active (Walterová et al., 1995).

Recently, the toxicity of SA and CHE in rat hepatocytes (Ulrichová et al., 1996) and cytotoxic effects of SA in cultured human fibroblasts (Karjalainen et al., 1988) were described. More studies on the molecular level are needed to clarify the adverse effects of both alkaloids on cells in vitro, and to expand our knowledge about potential toxicity in vivo. Primary human hepatocytes are currently considered to be an appropriate model for the assessment of safety of pharmaceutical and nutritional products. Furthermore, the studied compounds are a part of a veterinary preparation for pigs. Therefore, in this study, both human and porcine hepatocytes were used.

Section snippets

Alkaloids and chemicals

Sanguiritrine, a QBA extract from Macleaya cordata, containing 71% QBA in ratio SA:CHE:minor QBA (6.3:2.4:1.3, determined by HPLC) was purchased from CAMAS Technologies, Inc. (Broomfield, USA). Sanguinarine and chelerythrine were isolated from sanguiritrine using column chromatography on alumina (Dostál et al., 1992). Sanguinarine in 98.1% purity, MP 279–282 °C ((Southon and Buckingham, 1989) 277–280 °C) and chelerythrine in 95% purity, MP 200–204 °C ((Southon and Buckingham, 1989) 202–203 °C) were

Results

The addition of alkaloids SA and CHE to primary cultures of human and porcine hepatocytes resulted in time- and dose-dependent cell death as monitored by LDH leakage, GSH depletion and decrease of mitochondrial dehydrogenase activity (Section 2.5), while FA did not affect cell viability and functionality by any of the parameters mentioned above (Table 1). Similar results were obtained with rat hepatocytes (Ulrichová et al., 1996). SA was more potent than CHE in hepatocytes from all three

Discussion

One of the prerequisites for QBA biological activity is the presence of an iminium bond. In SA and CHE, this bond is susceptible to a nucleophilic attack and consequently plays a key role in the inhibition of SH-proteins. Both alkaloids interconvert between the cationic vs neutral form (i.e. hydroxide adduct or pseudobase) displaying sort of ‘Dr Jekyll and Hyde’ duality. They penetrate across the cell membrane in the hydrophobic pseudobase form acting as the pro-drugs and convert into the

Acknowledgements

The financial support of the Grant Agency (311/98/0648), the Grant Agency of the Ministry of Health (NL 5267-3/1999), and Ministry of Education of Czech Republic (MSM 151100003) is greatly acknowledged.

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