Development of an in vitro screening method of acute cytotoxicity of the pyrrolizidine alkaloid lasiocarpine in human and rodent hepatic cell lines by increasing susceptibility

Kristina Forsch, Verena Schöning, Lucia Disch,Beate Siewert, Matthias Unger, Jürgen Drewe

Abstract

Ethnopharmacological relevance
Pyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential.
Aim of the study
The aim was to develop an in vitro screening method of their cytotoxicity.
Materials and Methods
Human and rodent hepatocyte cell lines (HepG2 and H-4-II-E) were used to assess cytotoxicity of the PA lasiocarpine. At concentrations of 25 µM up to even 2400 µM, no toxic effects in neither cell line was observed with standard cell culture media. Therefore, different approaches were investigated to enhance the susceptibility of cells to PA toxicity (using high-glucose or galactose-based media, induction of toxifying cytochromes, inhibition of metabolic carboxylesterase, and inhibition of glutathione-mediated detoxification).
Results
Galactose-based culture medium (11.1 mM) increased cell susceptibility in both cell-lines. Cytochrome P450-induction by rifampicin showed no effect. Inhibition of carboxylesterase-mediated PA detoxification by specific carboxylesterase 2 inhibitor loperamide (2.5 µM) enhanced lasiocarpine toxicity, whereas the unspecific carboxylesterase inhibitor bis(4-nitrophenyl)phosphate (BNPP, 100 µM)) had a weaker effect. Finally, the inhibition of glutathione-mediated detoxification by buthionine sulphoximine (BSO, 100 µM) strongly enhanced lasiocarpine toxicity in H-4-II-E cells in low and medium, but not in high concentrations.
Conclusions
If no toxicity is observed under standard conditions, susceptibility enhancement by using galactose-based media, loperamide, and BSO may be useful to assess relative acute cytotoxicity of PAs in different cell lines.

Keywords: pyrrolizidine alkaloids; cytotoxicity, in vitro, H-4-II-E cells; HepG2 cells Chemical compounds studied in this article:

1 Introduction

Pyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators (Hartmann and Witte, 1995; Langel et al., 2011). PAs, ester alkaloids composed of a necine base (two fused five-membered rings joined by a single nitrogen atom) and a necic acid (one or two carboxylic ester arms) are generally considered acutely hepatotoxic, genotoxic and carcinogenic. The main organ of PA metabolism and target of toxicological effects is the liver (Bull and Dick, 1959; Bull et al., 1958; Butler et al., 1970; DeLeve et al., 1996; Jago, 1971; Li et al., 2011; Neumann et al., 2015). Three principal metabolic pathways for 1,2-unsaturated PAs of the retronecine-type are known (Chen et al., 2010): (i) Detoxification: Hydrolysis of the C7/ C9 ester bond by non-specific esterases to release necine base and necic acid. These intermediates are then subjected to further phase II-conjugation and excretion. (ii) Detoxification: N-oxidation of the necine base to form PA N-oxides. (iii) Metabolic activation/ toxification: Oxidation and/or oxidative N-demethylation, resulting after cleaving the ester bond(s) by esterases in the formation of reactive pyrroles (also known as dehydropyrrolizidine or pyrrolic ester) (Figure 1). This pathway is mainly catalysed by hepatic cytochrome P450 (CYP) isoforms CYP2B and 3A (Ruan et al., 2014). Reactive pyrroles cause damage in the cells in which they are formed, usually hepatocytes, but can pass from the hepatocytes into the adjacent sinusoids and damage the endothelial lining (Gao et al., 2015) mainly by reaction with DNA, protein, and lipids. Due to the ability of 1,2-unsaturated PAs to form DNA adducts, DNA crosslinks and DNA breaks, they are generally considered genotoxic and carcinogenic (Chen et al., 2010; EFSA, 2011; Fu et al., 2004; Li et al., 2011; Takanashi et al., 1980; Yan et al., 2008; Zhao et al., 2012). After acute intoxication of humans, the most common lesions in the liver are haemorrhagic necrosis, lesions in the central and sublobular veins of the liver, and acute veno-occlusive disease (DeLeve et al., 2003; EFSA, 2011).
However, up until to now, only few in vitro and in vivo investigations were performed to evaluate the relative toxic potential of PAs (Field et al., 2015; Li et al., 2011; Tamta et al., 2012). Especially in in vitro studies, the susceptibility of different cells lines to acute toxic effects of PAs was low (Field et al., 2015), which further complicates those kind of studies. This could be due to the fact that some cell lines switch their metabolism according to the so-called Crabtree effect (Crabtree, 1928). This effect describes that in presence of low-glucose concentrations cells in culture derive all their energy from anaerobic glycolysis rather than via mitochondrial oxidative phosphorylation (OXPHOS) despite of aerobic conditions. This leads to a high resistance against mitochondrial toxins (Marroquin et al., 2007). Mitochondrial toxicity, which may be related to the acute toxicity of PAs, was shown among others by for the PAs clivorine and senecionine (Ji et al., 2008), retrorsine (Gordon et al., 2000), lasiocarpine (Armstrong et al., 1972) and dehydromonocrotaline (Mingatto et al., 2007).
On this account the aim of this study was to develop a suitable screening system by reducing the threshold of susceptibility to toxic effects. The acute toxic effects of PAs were then studied in those sensitized cells. This included a general approach of modification of the cell culture medium, and specific alterations to the activity of enzymes, which are involved in the metabolism of PAs.

2 Materials and Methods

2.1 Chemical and reagents

Lasiocarpine, was obtained from Phytolab (Vestenbergsgreuth, Germany). The specific carboxylesterase (CES) 2 inhibitor loperamide hydrochloride, the unspecific CES inhibitor bis(4-nitrophenyl)phosphate (BNPP), DL-buthionine sulphoximine (BSO) and rifampicin were purchased from Sigma Aldrich (St. Louis, Missouri, USA) in the highest grade available. Media MEM-Glutamax media, DMEM-Glutamax media, sodium pyruvate, MEM non-essential amino acids, penicillin/streptomycin, L-glutamine, 2-(4-(2hydroxyethyl)-1-piperazinyl)-ethansulphoic acid (HEPES) and foetal bovine serum (FBS) were obtained from Gibco (Carlsbad, Californian, USA). The positive control digitonin was purchased from Sigma Aldrich (St. Louis, Missouri, USA). WST-1 kit was purchased from BioVision (Milpitas, California, USA).

2.2 Cells

The human hepatocellular carcinoma (HepG2) and the rat hepatocellular carcinoma (H-4II-E) cell lines, purchased from ATCC (LGC Standards, Middlesex TW11 0LY, UK) were maintained in 75 cm2 culture flask (Semadeni, Ostermundigen, CH) as adherent cell lines in low-glucose MEM-Glutamax (5.5 mM D-glucose) media with 10% (V/V) FBS, 0.5 mM sodium pyruvate, MEM non-essential amino acids and 1% (V/V) penicillin/ streptomycin. The high-glucose DMEM-Glutamax media (25 mM D-glucose) consisted of 10% (V/V) FBS, 0.5 mM sodium pyruvate, and MEM non-essential amino acids. The galactose-based DMEM-Glutamax media (without D-glucose) consisted of 10% (V/V) FBS, 0.5 mM sodium pyruvate, MEM non-essential amino acids, 11.1 mM galactose, 1% (V/V) L-glutamine and 0.5% (V/V) HEPES. The values of glucose or galactose were defined as the glucose or galactose concentration in the media, not in the cells. Galactose-based DMEM-Glutamax media were used to prevent the energy production via glycolysis in the cultivated cells (Crabtree effect (Crabtree, 1928)).

2.3 Treatment conditions

2.3.1 PA toxicity without pre-treatment

H-4-II-E and HepG2 cells were differentiated for 72 h and then the cells were incubated in low-glucose (5.5 mM) media with lasiocarpine up to 2400 µM every 24 h for further three days. This experiment was entirely performed in low-glucose (5.5 mM) media.

2.3.2 Induction of cytochromes

H-4-II-E and HepG2 cells were differentiated for 72 h. Then the cells were induced every 24 h for three days with rifampicin (25 µM) to increase cytochrome expression and activity. Afterwards, the cells were additionally incubated with lasiocarpine up to 900 µM and rifampicin every 24 h for further three days. This experiment was entirely performed in low-glucose (5.5 mM) media.

2.3.3 Change in media

To increase susceptibility, H-4-II-E cells were cultured in two different media: (1) Cells were cultivated and differentiated for 24 h and treated with lasiocarpine every 24 h for three days in high-glucose-based media (25 mM D-glucose). (2) Cells were cultivated and differentiated over 24 h in high-glucose (25 mM D-glucose) based media and then switched to galactose-based medium (11.1 mM) 24 h prior to and during the lasiocarpine treatment. Cells were treated with lasiocarpine every 24 h for three days.

2.3.4 Inhibition of detoxification pathways

2.3.4.1 Inhibition of carboxylesterases

To increase susceptibility in H-4-II-E and HepG2 cells, carboxylesterases (CES), which are involved in the detoxification of PAs, were inhibited. Cells were cultivated and differentiated for 24 h in high-glucose-based media (25 mM D-glucose), and then switched to galactose-based medium (11.1 mM) 24 h prior to treatment. Cells were treated every 24 h for three days with (1) lasiocarpine up to 900 µM and the unspecific CES inhibitor BNPP (100 µM) or (2) lasiocarpine up to 900 µM and the the specific CES-2 inhibitor loperamide (2.5 µM).

2.3.4.2 Inhibition of GSH formation

The detoxification of lasiocarpine was inhibited by reducing the glutathione synthesis with BSO), an inhibitor of γ-glutamylcysteine synthetase (γ-GCS). BSO lowers tissue glutathione (GSH) concentrations. Cells were cultivated and differentiated for 24 h in high-glucose-based media (25 mM D-glucose), and then switched to galactose-based medium (11.1 mM) 24 h prior to treatment. Cells were treated every 24 h for three days with lasiocarpine up to 900 µM and BSO (100 µM).

2.4 WST-1 assay

The WST-1 test was used to measure the metabolic activity in the two cell lines (H-4-II-E and HepG2) as a marker for cellular toxicity. The toxicity was defined as decrease of metabolic activity of ≥ 20%. At the end of the experiment, 10 µL WST-1 reagent was added to each well. Plates were then incubated for 4 h to allow for the reduction of WST-1 reagent. Absorbance was measured by the microplate absorbance reader (Infinite M 200, Tecan Trading Ltd., Männedorf, CH) at 450 nm, reference wavelength of 620 nm (n = 3). The validity of the WST-1 assay was verified using digitonin (100 µM) as positive control and the metabolic activity was reduced to 0.9 – 12.5%.

3 Results

3.1 Susceptibility of cells to PAs without pre-treatment

The susceptibility of immortalized cell lines to toxic effect of lasiocarpine was examined in H-4-II-E and HepG2 cells after 72 h incubation without any pre-treatment in lowglucose media. Lasiocarpine was applied in doses of 25 µM to up to excessive concentrations of 2400 µM. The toxic effect of lasiocarpine was measured as decrease in metabolic activity evaluated by WST-1 assay. At the highest concentration, the metabolic activity decreased in both cell lines. The effect was more pronounced in H-4-II-E than in HepG2 cells (Figure 2).

3.2 Enhancement of susceptibility by induction of metabolic activation (rifampicin)

The induction of cytochromes did not increase the susceptibility of H-4-II-E und HepG2 cells to lasiocarpine toxicity (Figure 3).

3.3 Enhancement of susceptibility by changes in the medium (high-glucose versus galactose)

As H-4-II-E cells were more susceptible to lasiocarpine toxicity, the influence of the media was investigated in this cell line. A toxic effect as decrease in metabolic activity is observed in galactose and the high-glucose approach at lasiocarpine concentrations of 600 µM. The effect was more pronounced in cells treated in galactose media than in highglucose media (Figure 4). However, high-glucose media did also increase the susceptibility to lasiocarpine toxicity of H-4-II-E cells compared to the first experiment in low-glucose media (5.5 mM D-glucose).

3.4 Enhancement of susceptibility by inhibition of detoxification (carboxylesterases and glutathione formation)

3.4.1 Inhibition of carboxylesterases (CES)

A further approach to increase susceptibility of immortalized cells to toxic effects of PAs is to increase the number of reactive pyrroles by inhibition of their detoxification pathways. Treatment with both CES-inhibitors led to a decrease in the metabolic activity. At the two highest concentrations of lasiocarpine (600 µM and 900 µM) with loperamide, a reduction in metabolic activity down to 59 and 38% in H-4-II-E cells, and 66 and 49% in HepG2 cells, respectively, was observed (Figure 5 and Figure 6).
Furthermore, treatment with lasiocarpine in combination with the unspecific CES inhibitor BNPP also reduced, but less effective, metabolic activity. At the highest concentration of 900 µM, lasiocarpine reduced metabolic activity down to 56 and 73% in H-4-II-E and HepG2 cells, respectively. Loperamide alone had no effect on the metabolic activity (H-4-II-E: 120.4% and HepG2: 91.0%) of both cell lines.

3.4.2 Inhibition of glutathione formation

Inhibition of glutathione synthesis with BSO (100 µM) revealed a strong decrease in the metabolic activity of H-4-II-E at the low and medium concentrations of lasiocarpine (50300 µM). However, at higher concentrations (600 µM and 900 µM), the metabolic activity increased to 126% (Figure 7). The visual control of the cells (data not shown) also confirmed this result: at low and medium concentrations, the cell density was lower and gaps in the monolayer were visible; at the highest concentration, the cell density was comparable with solvent control and the monolayer was confluent. In contrast, the inhibition of glutathione in HepG2 cells did not show any effect on the metabolic activity (Figure 8).

4 Discussion

The use of primary human hepatocytes is to date the standard in vitro model to investigate cytotoxicity. But these cells are very cost-intensive and difficult to handle. Human and rodent hepatic immortalized cell lines are alternative systems, which are easier in handling and cost-effective. The human hepatic cell line HepG2 was used for many investigations of cytotoxic compounds including PA toxicity (Li et al., 2013; Tamta et al., 2012). These studies revealed that for a suitable and well-established in vitro screening system for PA toxicity, the susceptibility of the cells needs to be increased.
Different alterations, e.g. by induction or inhibition of metabolic pathways in immortalized human HepG2 and rodent H-4-II-E cells, were evaluated with regard to their influence on the cytotoxicity of the PA lasiocarpine using WST-1 assay. The general assumption implies that cytotoxicity of PAs primary depends on their metabolic activation by CYP enzymes to form reactive pyrroles, which leads to covalent adduct formation with cellular nucleophiles (Li et al., 2013). In the first experiment, we examined the susceptibility of cells to lasiocarpine toxicity up to excessively high concentrations of 2400 µM without any pre-treatment. The results revealed a decrease in metabolic activity compared to the solvent in both cell lines at the highest concentration only. This effect was more pronounced in H-4-II-E cells than in HepG2 cells. This result suggests that H-4-II-E cells may have a higher metabolic activity than HepG2 cells, which leads to a higher metabolic toxification of toxic pyrroles and thus increased susceptibility. However, both cell lines can be considered as resistant to lasiocarpine toxicity.
Induction of CYP enzyme activity with rifampicin prior to treatment did not increase susceptibility for lasiocarpine toxicity in both cell lines. This could be due to two different reasons: (1) rifampicin: this substance is known to also induce phase-II enzymes (Doostdar et al., 1993; Westerink and Schoonen, 2007), which would increase the detoxification of the reactive pyrroles. (2) the Crabtree effect (Aguer et al., 2011; Marroquin et al., 2007): in this case, although cultured under aerobic conditions, cell lines with low supply of glucose metabolically rely on anaerobic glycolysis rather than mitochondrial OXPHOS to generate the required energy. This phenomenon increases the resistance to toxic effects of many mitochondrial function impairing drugs.
Therefore, in the third experiment, we successfully tried to circumvent the Crabtree effect with two different approaches in H-4-II-E cells: (1) Switching the cells from a highglucose media (cultivation) to a glucose-free, galactose-based media during treatment (Iyer et al., 2010). (2) Cultivation and treatment in high-glucose media, as high concentrations of glucose inhibit the hexokinase (which is an important enzyme in the glycolytic pathway) (Marin-Hernandez et al., 2006). Toxic effects were already seen at moderate lasiocarpine concentrations of 600 µM in both approaches, but the galactosebased media resulted in a higher increase in the susceptibility to PAs.
A further approach of our study was the inhibition of the detoxification pathways including hydrolysis by carboxylesterases and glutathione conjugation of reactive pyrroles. Our present findings demonstrated that the inhibition of carboxylesterases by loperamide and BNPP leads to a significant decrease in metabolic activity in H-4-II-E cells and HepG2 cells, with the specific inhibitor loperamide being more effective. Therefore, carboxylesterase activity has a strong impact on the detoxification of PAs and consequently on the susceptibility of the cells.
The inhibition of glutathione synthesis by BSO in H-4-II-E cells resulted in a strong decrease in metabolic activity at low and medium concentrations and an increase at the two highest concentrations. Cytotoxicity is a complex interplay of several physiological mechanisms. One possible explanation for this observed hormetic effect may be the induction of phase-II enzymes, e.g. UDP-glucuronosyltransferase, by oxidative stress at high concentrations (Kalthoff et al., 2010), which would then increase the detoxification of the reactive pyrroles. For the PA senecionine it was shown, that UDP-glucuronosyltransferase 1A4 is involved in its detoxification (He et al., 2010). However, the investigation of the exact reason for this phenomenon is out of scope of this study and will be investigated separately.
In HepG2 cells, inhibition of glutathione synthesis did not lead to a change in metabolic activity. This is due to the lower CYP enzyme activity in this cell line, and therefore a lower toxification of lasiocarpine to reactive pyrroles, and lower toxic effects (Kalthoff et al., 2010; Westerink and Schoonen, 2007).

5 Conclusions

In the present in vitro study, the susceptibility to lasiocarpine in HepG2 and H-4-II-E cells under different conditions was investigated. Inhibition of glycolysis by treating the cells in galactose-based media and inhibition of carboxylesterases increased the susceptibility of both cell lines, whereas the inhibition of GSH was only suitable in H-4-II-E cells. Especially, the former two approaches provide a useful method to perform in vitro screening of PA toxicity in immortalized cell lines. Furthermore, this study emphasises the necessity to proof the suitability and susceptibility of the in vitro test system before assessing compound toxicity.

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