Assessment of Hepatotoxic Potential of Cyanobacterial Toxins Using

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Ecotoxicology and Human Environmental Health

Assessment of hepatotoxic potential of cyanobacterial toxins using 3D in vitro model of adult human liver stem cells Amrita Basu, Aneta Dydowiczova, Lucie Ctverackova, Libor Jasa, James Edward Trosko, Ludek Blaha, and Pavel Babica Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02291 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Environmental Science & Technology

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Assessment of hepatotoxic potential of cyanobacterial toxins using 3D in vitro model of adult human

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liver stem cells

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Amrita Basua, Aneta Dydowiczováa, Lucie Čtveráčkováa, Libor Jašaa, James E. Troskob, Luděk Bláhaa,

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Pavel Babicaa*

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a

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00, Czech Republic

RECETOX, Faculty of Science, Masaryk University, Masaryk University, Kamenice 753/5, Brno, 625

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b

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State University, 1129 Farm Lane, East Lansing, 48824, MI, USA

Department of Pediatrics and Human Development & Institute for Integrative Toxicology, Michigan

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*Corresponding author:

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Pavel Babica

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RECETOX

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Faculty of Science

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Masaryk University

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Kamenice 753/5, building A29

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625 00

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Brno

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Czech Republic

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Email: [email protected]

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Tel: (+420) 549 494 620

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Fax: (+420) 549 49 2840

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ORCID: P.B. 0000-0001-6399-3554

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ACS Paragon Plus Environment

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Abstract Art

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Abstract

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Cyanotoxins microcystin-LR (MC-LR) and cylindrospermopsin (CYN) represent hazardous waterborne

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contaminants and potent human hepatotoxins. However, in vitro monolayer cultures of hepatic cell lines

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were found to recapitulate, poorly, major hepatocyte-specific functions and inadequately predict

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hepatotoxic effects of MC-LR and CYN. We utilized 3-dimensional (3D), scaffold-free spheroid cultures

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of human telomerase-immortalized adult liver stem cells HL1-hT1 to evaluate hepatotoxic potential of

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MC-LR and CYN. In monolayer cultures of HL1-hT1 cells, MC-LR did not induce cytotoxic effects (EC50

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> 10 micromole/L), while CYN inhibited cell growth and viability (48h-96h EC50 ~5.5-0.5 micromole/L).

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Growth and viability of small growing spheroids were inhibited by both cyanotoxins (≥0.1 micromole/L)

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and were associated with blebbing and disintegration at the spheroid surface. Hepatospheroid damage and

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viability reduction were observed also in large mature spheroids, with viability 96h-EC50 values being

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0.04 micromole/L for MC-LR and 0.1 micromole/L for CYN, and No Observed Effect Concentrations

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7.7 µM (24 h), 0.14-0.48 µM (48 h), and 0.1 µM

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(72 h)

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concentrations (0.01-0.1 µM) of CYN. As indicated by the results of real-time impedimetric analysis in

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monolayers of HL1-hT1 cells, the inhibition of growth and viability started after 24 h of exposure and

25–27

, C3A

or HepaRG

, where

. Unlike monolayers, spheroid cultures of HL1-hT1 cells were sensitive to such low


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became more pronounced further, which is in agreement with other studies indicating a steep increase in

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CYN cytotoxicity from 24 h to 48-72 h of exposure 25,37,38. The lack of apparent cytotoxic effect during the

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first 24 h of monolayer exposure could also explain the absence of CYN effects on the formation of

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spheroid from monolayer-harvested cells. However, CYN effects on spheroids progressed in time, and the

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toxin caused spheroid damage in large 168h-old mature spheroids, which indicates that HL1-hT1 spheroid

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cultures maintained their sensitivity to CYN cytotoxicity during the long-time culture. Cytochrome P450

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(CYP450)-dependent bioactivation might play a role in CYN hepatotoxicity and cytotoxicity, as different

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modulators of CYP450 were found to alter CYN effects 1,2,5,6. Moreover, differentiated HepaRG cells with

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higher CYP450 activity were found to be more sensitive to CYN than undifferentiated cells

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monolayer cultures of HL1-hT1 cells do not express major hepatocyte markers, induction of CYP450

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genes, in response to xenobiotics and drugs, was previously observed in both parental HL1-1 and

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immortalized HL1-hT1 cells

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differentiation in HL1-hT1 cells could be further stimulated by 3D culture conditions, as liver-specific

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functions, including CYP450 activity, are known to be induced in spheroid cultures of other hepatic cell

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lines, such as HepG2 or HepaRG

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transformation of CYN in differentiated, metabolically-competent and CYN-sensitive HepaRG cells

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Correspondingly, we also did not observe any significant depletion of CYN concentrations in the culture

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medium of HL1-hT1 cells affected by CYN cytotoxicity. Thus, the role of CYP450 in CYN toxicity needs

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to be further examined in future research, where hepatospheroid cultures could provide a sensitive tool to

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study mechanisms of CYN cytotoxicity and bioactivation.

56,67,68

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. While

. Expression of biotransformation enzymes and induction of hepatic

44–46

. However, Kittler et al. (2016) did not detect CYP450-dependent 42

.

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Although majority of in vitro studies traditionally relies on the nominal concentrations to define

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concentration-effect relationships, the real bioavailable concentrations of the tested toxicants within a

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given in vitro system might substantially differ from the nominal concentrations due to a variety of

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factors, including binding of the chemical to the plastic materials, interactions with the medium or cell

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components, evaporation, degradation or biotransformation of the chemical during the in vitro experiment

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69

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laboratory handling and storage

. In fact, MC-LR has been reported to adsorb to the lab ware and to be subject of various losses during 70,71

, and both cyanotoxins might be biotransformed during the in vitro

1

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experiments . These issues could have become more exacerbated in our spheroid experiments, which

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included longer exposure times (up to 2 weeks), multiple exchanges of the exposure medium, and possible

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interactions with the agarose hydrogel. However, LC-MS/MS analysis revealed only minor and

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toxicologically most likely insignificant fluctuations of the total cyanotoxin concentrations in the exposure

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medium, which were mostly associated with the medium exchanges and gel equilibration during the

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spheroid experiments. Concentrations of cyanotoxins were unaffected by the presence of the cells, thus the

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extent of eventual toxin uptake or biotransformation did not cause their detectable depletion in the


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exposure medium, which is in agreement with recent reports for CYN 42. Overall, total concentrations of

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cyanotoxins were maintained at the desired levels throughout the course of in vitro exposures, which

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provides an additional quality control for the toxicological experiments and assures more reliable

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interpretation of the in vitro data.

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Our study represents probably the first example of 3D in vitro model, utilizing a permanent

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continuous adult liver stem cell line, which was capable to detect hepatotoxic potential of both MC-LR

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and CYN at relatively low concentrations (0.01-0.1 µM). These effective concentrations were comparable

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not only with concentrations inducing cytotoxicity in primary liver cells in vitro

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concentrations found in blood serum (MC-LR: 0.01-0.06 µM) or liver tissue (MC-LR: 0.01-2 µM, CYN:

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0.002-0.3 µM) of animals experimentally exposed to sublethal cyanotoxin doses 9–13, or in liver tissues of

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MC-intoxicated humans with a fatal or transplant-requiring liver failure (0.05-0.5 µM)

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weight ratio of 1, and fresh: dry weight ratio of 5, were used to recalculate liver tissue concentrations). In

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vitro detection of cyanotoxin toxicity with such sensitivity has required so far either the use of primary

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liver cells or different approaches to increase the sensitivity of permanent cell lines, such as

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overexpression of OATP transporters

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of CYP450 43. In contrast, 3D in vitro model based on human adult liver stem cells allows to study toxicity

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of low cyanotoxin concentrations, but in a physiologically more relevant setup

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limited availability, limited life-span and variable quality of human primary liver cells, and without the

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need to rely on either rodent hepatocytes, or cancer-derived human hepatic cell lines with their inherent

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(epi-)genetic abnormalities

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like sensitivity to cyanotoxins even for extended periods of time (96-336 h), allowing to study long-term

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effects of even lower, subcytotoxic cyanotoxin levels, since MCs (0.1-2 nM) have been detected in blood

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of human populations chronically exposed to cyanobacteria and associated with various adverse outcomes,

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such hepatocellular damage 16,17, metabolic diseases 18 and liver cancer 8.

44–46

57,72,73

, membrane permeabilization

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19–27

, but also to

14,15

(volume:

or pharmacological induction 75,76

, while bypassing the

. Moreover, spheroids of HL1-hT1 cells acquired and maintained in vivo-

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Occurrence of toxic cyanobacterial blooms in fresh and coastal waters represents a global

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environmental issue, which might become even more serious due to rising human population and

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increasing demands for clean water resources

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ecological and human health risks of cyanotoxins are therefore needed, including sensitive in vitro models

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to detect and study cyanotoxin effects at environmentally or toxicologically relevant concentrations. HL1-

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hT1 hepatospheroids were produced using a commercially available, scaffold-free micromolding system,

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which can be repeatedly and nearly indefinitely used. Size-defined spheroids can be prepared without a

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need for an additional instrumentation (e.g. bioreactors), special skills (e.g. soft-lithography) or specific

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single-use cultureware (hanging drop plates, low attachment plates). This technique can be easily

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introduced into any laboratory equipped for basic in vitro cell culture work, and it can be eventually used

1,2

. Effective tools for assessment and management of


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also in combination with other hepatic cell lines 54, such as HepG2 or HepaRG, whose hepatocyte-specific

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characteristic are known to improve in 3D cultures 44–46. Therefore, such 3D in vitro liver model provides

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a simple and practical tool, which can be employed not only in environmental and toxicological research

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of cyanotoxins or other environmental hepatotoxins, but also in effect-based assessment of cyanobacteria-

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or cyanotoxin contaminated samples. These samples represent complex mixtures of chemicals, which

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might not be fully chemically and toxicologically characterized

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occurring in hundreds of structural variants, 2) other bioactive cyanobacterial metabolites, 3) products and

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by-products of natural or drinking water treatment-related transformation or degradation of cyanotoxins,

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4) other contaminants. Thus, evaluation of hepatotoxic potential using a sensitive 3D human liver in vitro

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assay could be an effective approach providing information complementary to the chemical analyses,

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which can be utilized for an integrated chemical-biological effect assessment and monitoring of

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recreational and drinking water quality and safety, and for an improved assessment of human health risks

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of toxic cyanobacteria.

1–3

, including: 1) different cyanotoxins

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Conflict of Interest: The authors declare that they have no conflict of interest.

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Acknowledgement: We thank Ms. Sabina Váňová and Ms. Hana Klímová for their technical help and

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assistance with the experiments. Research was supported by Czech Science Foundation (no. GA15-

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12408S), and infrastructural projects from the Czech Ministry of Education, Youth and Sports (grant

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numbers LM2015051 and CZ.02.1.01/0.0/0.0/16_013/0001761).

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Supporting Information. Method information on cell culture techniques, viability assays and cyanotoxin

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analysis; figures showing cyanotoxin effects in monolayer cultures (cell impedance, viability,

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microphotographs), spheroid cultures (spheroid growth and microphotographs), results of cyanotoxin

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analysis; a table summarizing NOEC/LOEC/EC25/EC50 values.

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Figure Legends

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Figure 1. Effects of microcystin-LR (MC-LR) and cylindrospermopsin (CYN) on the growth and

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viability of adult human liver stem cells in monolayer experiments

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HL1-hT1 cells were treated 48 h post-seeding by MC-LR or CYN. (A) Cell growth and condition were

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monitored by impedimetric analysis for 120 h of exposure, the results are expressed as control-normalized

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Delta Cell Index values. (B) Cell viability was evaluated by Alamar Blue assay after 96 h exposure. NT -

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non-treated control (Control), Solvent - solvent control (0.2% MeOH, v/v). Data represent average±SD

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values from independently repeated experiments, the curves represent a fit of 3-parameter sigmoid

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function. *Data points below the dashed line were significantly lower (Mann-Whitney test, p10 n.a. n.a. 2.91 n.a. n.a.

>10 n.a. n.a. 0.62 n.a. n.a.

250 cell/spheroid Relative Alamar Spheroid Blue Growth n.a. d 2.15 n.a. 0.39 0.16 0.15 n.a. 0.38 n.a. 0.14 0.12 0.12

a

4000 cell/spheroid Relative Alamar Spheroid Blue Growth >10 0.04 >10 0.08 n.a. n.a. >2.5 0.10 >2.5 0.05 n.a. n.a.

EC50 values were estimated using a 3-parameter sigmoid function In monolayer experiments, cell growth and viability were evaluated using Neutral Red Uptake (NRU), Alamar Blue and CFDA-AM assays as well as using impedimetric analysis (%DCI – control-normalized Delta Cell Index) c In spheroid experiments, EC50 values were calculated from control-normalized spheroid growth (Relative Spheroid Growth) and from viability assay (Alamar Blue). d n.a. – not available, the condition was not tested b

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FIGURE 1 705 A)

B)


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FIGURE 2 A)

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C)

D)


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FIGURE 3 A)

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B)

C)


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FIGURE 4 2 A)

B)


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Assessment of Hepatotoxic Potential of Cyanobacterial Toxins Using

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