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. 2023 Jan 1:249:114439.
doi: 10.1016/j.ecoenv.2022.114439. Epub 2022 Dec 19.

Potential for cardiac toxicity with methylimidazolium ionic liquids

Tarek M Abdelghany  1 , Shireen A Hedya  2 , Carol De Santis  3 , Sahar S Abd El-Rahman  4 , Jason H Gill  3 , Noha F Abdelkader  5 , Matthew C Wright  6
Affiliations

Potential for cardiac toxicity with methylimidazolium ionic liquids

Tarek M Abdelghany et al. Ecotoxicol Environ Saf. .

Abstract

Methylimidazolium ionic liquids (MILs) are solvent chemicals used in industry. Recent work suggests that MILs are beginning to contaminate the environment and lead to exposure in the general population. In this study, the potential for MILs to cause cardiac toxicity has been examined. The effects of 5 chloride MIL salts possessing increasing alkyl chain lengths (2 C, EMI; 4 C, BMI; 6 C; HMI, 8 C, M8OI; 10 C, DMI) on rat neonatal cardiomyocyte beat rate, beat amplitude and cell survival were initially examined. Increasing alkyl chain length resulted in increasing adverse effects, with effects seen at 10-5 M at all endpoints with M8OI and DMI, the lowest concentration tested. A limited sub-acute toxicity study in rats identified potential cardiotoxic effects with longer chain MILs (HMI, M8OI and DMI) based on clinical chemistry. A 5 month oral/drinking water study with these MILs confirmed cardiotoxicity based on histopathology and clinical chemistry endpoints. Since previous studies in mice did not identify the heart as a target organ, the likely cause of the species difference was investigated. qRT-PCR and Western blotting identified a marked higher expression of p-glycoprotein-3 (also known as ABCB4 or MDR2) and the breast cancer related protein transporter BCRP (also known as ABCG2) in mouse, compared to rat heart. Addition of the BCRP inhibitor Ko143 - but not the p-glycoproteins inhibitor cyclosporin A - increased mouse cardiomyocyte HL-1 cell sensitivity to longer chain MILs to a limited extent. MILs therefore have a potential for cardiotoxicity in rats. Mice may be less sensitive to cardiotoxicity from MILs due in part, to increased excretion via higher levels of cardiac BCRP expression and/or function. MILs alone, therefore may represent a hazard in man in the future, particularly if use levels increase. The impact that MILs exposure has on sensitivity to cardiotoxic drugs, heart disease and other chronic diseases is unknown.

Keywords: AR42J-B13; C8[mim]; Heart; Ionic liquids; MTT; Transporter.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Effect of EMI on rat cardiomyocyte function and viability in vitro. A, representative traces showing changes in cell contractility following exposure to EMI at the indicated concentration and timepoints after addition. B, changes in beat rate and beat amplitude are represented as fold changes relative to control. Results are the mean of at least 3 separate determinations from the same experiment ± SD, *p < 0.05. C, xCELLigence trace showing cell index (indicative of cell survival) over time. Data plot is normalised to point of compound addition. All results typical of at least 3 separate experiments.
Fig. 2
Fig. 2
Effect of BMI on rat cardiomyocyte function and viability in vitro. A, representative traces showing changes in cell contractility following exposure to BMI at the indicated concentration and timepoints after addition. B, changes in beat rate and beat amplitude are represented as fold change relative to control. Results are the mean of at least 3 separate determinations from the same experiment ± SD, *p < 0.05. C, xCELLigence trace showing cell index (indicative of cell survival) over time. Data plot is normalised to point of compound addition. All results typical of at least 3 separate experiments.
Fig. 3
Fig. 3
Effect of HMI on rat cardiomyocyte function and viability in vitro. A, representative traces showing changes in cell contractility following exposure to HMI at the indicated concentration and timepoints after addition. B, changes in beat rate and beat amplitude are represented as fold change relative to control. Results are the mean of at least 3 separate determinations from the same experiment ± SD, *p < 0.05. C, xCELLigence trace showing cell index (indicative of cell survival) over time. Data plot is normalised to point of compound addition. All results typical of at least 3 separate experiments.
Fig. 4
Fig. 4
Effect of M8OI and DMI on rat cardiomyocyte function and viability in vitro. A, representative traces showing changes in cell contractility following exposure to M8OI at the indicated concentration and timepoints after addition. B, xCELLigence trace for M8OI showing cell index (indicative of cell survival) over time. Data plot is normalised to point of compound addition. C, representative traces showing changes in cell contractility following exposure to DMI at the indicated concentration and timepoints after addition. D, xCELLigence trace for DMI showing cell index (indicative of cell survival) over time. Data plot is normalised to point of compound addition. All results typical of at least 3 separate experiments.
Fig. 5
Fig. 5
Effect of MILs on cardiac injury in rats. Adult male rats (3 per group) were treated daily by intraperitoneal injection with PBS (vehicle control) containing additionally the indicated MIL at the dose indicated. Blood samples were collected 6 h after the last dose. A) Serum troponin I and B) serum CK-MB levels at sacrifice. Data are the mean and SD of 3 animals per group. *Statistically significantly different (two tailed, p < 0.05) from control using the one way analysis of variance (ANOVA) test followed by Bonferroni multiple comparison’s tests. C, adult male rats (7 per group) were exposed to drinking water (control) containing additionally 880 mg/L of a MIL for 5 months, prior to sacrifice and examination of tissues: photomicrograph of H&E-stained heart sections of; (i) control rat showing normal histological structure, (ii and iii) HMI treated rat showing diffuse vacuolar degeneration (arrow) of the cardiac muscle fibres, scattered hypereosinophilia (dashed arrow), and congestion of the coronary blood vessels (Co); (iv and v) MOI treated rat showing cardiomyocyte degeneration and increased hypereosinophilia (arrow), foci of macrophages phagocytosing the debris of the necrotic fibres (dashed arrow), and congestion of the coronary vessels (Co); (vi-viii) DMI treated rat showing vacuolization of the myocardial muscle fibres (arrow), hyalinization (dashed arrow), aggregation of macrophages engulfing necrotic debris, congestion (Co), and coronary vessel wall hyalinization (blue arrow) and perivascular oedema (thin arrow). D, severity of histopathological lesions were scored by a pathologist blinded to the treatment groups using a scale of 0–4 where “0” denotes absence of the histopathological lesion in the examined heart section; “1” signalizes that the distribution of the observed lesion is up to 25% of the area examined; “2” signalizes that the distribution of the observed lesion is between 26% and 50% of the area examined; “3” signalizes that the distribution of the observed lesion is between 51% and 75% of the area examined; and “4” signalizes that the distribution of the observed lesion is more than 75% of the area examined. These non-parametric data are presented as median (max-min) and were analysed using the Kruskal Wallis H test followed by the Mann-Whitney U test. *Significantly different (two tailed, p < 0.05) when compared to control group; #significantly different (two tailed, p < 0.05) when compared to HMI group; $significantly different (two tailed, p < 0.05) when compared to MOI. E, serum troponin and F, serum CK-MB levels. Data are the mean and SD of 7 animals/group. *Significantly different (two tailed, p < 0.05) from control using the one way analysis of variance (ANOVA) test followed by Bonferroni multiple comparison’s tests.
Fig. 5
Fig. 5
Effect of MILs on cardiac injury in rats. Adult male rats (3 per group) were treated daily by intraperitoneal injection with PBS (vehicle control) containing additionally the indicated MIL at the dose indicated. Blood samples were collected 6 h after the last dose. A) Serum troponin I and B) serum CK-MB levels at sacrifice. Data are the mean and SD of 3 animals per group. *Statistically significantly different (two tailed, p < 0.05) from control using the one way analysis of variance (ANOVA) test followed by Bonferroni multiple comparison’s tests. C, adult male rats (7 per group) were exposed to drinking water (control) containing additionally 880 mg/L of a MIL for 5 months, prior to sacrifice and examination of tissues: photomicrograph of H&E-stained heart sections of; (i) control rat showing normal histological structure, (ii and iii) HMI treated rat showing diffuse vacuolar degeneration (arrow) of the cardiac muscle fibres, scattered hypereosinophilia (dashed arrow), and congestion of the coronary blood vessels (Co); (iv and v) MOI treated rat showing cardiomyocyte degeneration and increased hypereosinophilia (arrow), foci of macrophages phagocytosing the debris of the necrotic fibres (dashed arrow), and congestion of the coronary vessels (Co); (vi-viii) DMI treated rat showing vacuolization of the myocardial muscle fibres (arrow), hyalinization (dashed arrow), aggregation of macrophages engulfing necrotic debris, congestion (Co), and coronary vessel wall hyalinization (blue arrow) and perivascular oedema (thin arrow). D, severity of histopathological lesions were scored by a pathologist blinded to the treatment groups using a scale of 0–4 where “0” denotes absence of the histopathological lesion in the examined heart section; “1” signalizes that the distribution of the observed lesion is up to 25% of the area examined; “2” signalizes that the distribution of the observed lesion is between 26% and 50% of the area examined; “3” signalizes that the distribution of the observed lesion is between 51% and 75% of the area examined; and “4” signalizes that the distribution of the observed lesion is more than 75% of the area examined. These non-parametric data are presented as median (max-min) and were analysed using the Kruskal Wallis H test followed by the Mann-Whitney U test. *Significantly different (two tailed, p < 0.05) when compared to control group; #significantly different (two tailed, p < 0.05) when compared to HMI group; $significantly different (two tailed, p < 0.05) when compared to MOI. E, serum troponin and F, serum CK-MB levels. Data are the mean and SD of 7 animals/group. *Significantly different (two tailed, p < 0.05) from control using the one way analysis of variance (ANOVA) test followed by Bonferroni multiple comparison’s tests.
Fig. 6
Fig. 6
Mouse expresses higher levels of cardiac p-glycoprotein 3 and BCRP compared to rats. A, qRT-PCR quantification for the indicated transcript in rat and mouse tissues, expressed relative to the indicated normalised transcript indicated with $. Data are the mean and standard deviation tissue expression levels determined from 3 separate animals. *Statistically-significantly different fold (18S rRNA normalised) transcript level between rat and mouse using the Students T test (two tailed, p < 0.05). B, upper panel, Western blot for the indicated protein from 3 separate rat and mouse hearts; lower panel, relative expression levels of the indicated protein normalised to beta-actin. Full blot views are available in Figure S2. *Statistically-significantly different protein concentration (beta-actin normalised) between rat and mouse using the Students T test (two tailed, p < 0.05).
Fig. 6
Fig. 6
Mouse expresses higher levels of cardiac p-glycoprotein 3 and BCRP compared to rats. A, qRT-PCR quantification for the indicated transcript in rat and mouse tissues, expressed relative to the indicated normalised transcript indicated with $. Data are the mean and standard deviation tissue expression levels determined from 3 separate animals. *Statistically-significantly different fold (18S rRNA normalised) transcript level between rat and mouse using the Students T test (two tailed, p < 0.05). B, upper panel, Western blot for the indicated protein from 3 separate rat and mouse hearts; lower panel, relative expression levels of the indicated protein normalised to beta-actin. Full blot views are available in Figure S2. *Statistically-significantly different protein concentration (beta-actin normalised) between rat and mouse using the Students T test (two tailed, p < 0.05).
Fig. 7
Fig. 7
Mouse HL-1 cardiomyocytes express high levels of Bcrp/BCRP, are resistant to longer chain MIL toxicity and show increased sensitivity to longer chain MILs in the presence of the BCRP inhibitor Ko143. A, qRT-PCR quantification of Bcrp mRNA transcript in mouse heart and HL-1 cells. Data are the mean and standard deviation from 3 separate mouse hearts and 3 cultures of HL-1 cells. B, left panel, Western blot for the indicated protein from 3 separate mouse hearts and 3 cultures of HL-1 cells; right panel, expression levels of BCRP normalised to glyceraldehyde 3 phosphate dehydrogenase (GAPDH) levels. Note that the levels of immunoreactive beta-actin in HL-1 cells was very low and therefore could not be used to in quantification. The faster migrating protein in HL-1 cells is noted although the cause is not known but likely is dependent on altered glycosylation given that it is a membrane-associated protein. Full blot views are available in Figure S3. C, HL-1 cells were treated with increasing concentrations of the indicated MIL in the absence or presence of the indicated transporter inhibitor. MTT was determined 24 h later. Data are the mean and SD of 5individual determinations from the same experiment, typical of 3 separate experiments. *Significantly different (two tailed, p < 0.05) from control using the one-way analysis of variance (ANOVA) test followed by Bonferroni multiple comparison’s tests.
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