Assessment of acute and repeated pulmonary toxicities of oligo(2-(2-ethoxy)ethoxyethyl guanidium chloride in mice

Abstract

Oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) and polyhexamethyleneguanidine phosphate (PHMG-P) are cationic biocides containing a guanidine group. Direct exposure of the lungs to PHMG-P is known to induce pulmonary inflammation and fibrotic changes. Few studies have assessed the pulmonary toxicity of PGH, another member of the guanidine family. In this study, we assessed the acute and repeated toxicity of PGH and PHMG-P to compare the pathological progression induced by both chemicals. PGH (1.5 mg/kg) or PHMG (0.6 mg/kg) was instilled intratracheally to mice once or three times every 4 days; subsequently, cytokine levels were quantified and a histopathological examination was performed. To verify the toxic mechanism of PGH, we quantified cell viability and cytokine production induced by PGH or PHMG-P in the presence or absence of anionic material in cells. Instillation of PGH and PHMG-P into the mouse lung increased cytokine production, immune cell infiltration, and pulmonary fibrotic changes. These pathological changes were exacerbated over time in the single- and the repeated-dose PHMG-P groups, but were resolved over time in the PGH groups. PGH or PHMG-P showed cytotoxic effects, IL-1β secretion, and ROS production in a dose-dependent manner in human cell lines. However, the co-treatment of anionic materials with PGH or PHMG-P significantly reduced these toxic responses, which confirmed that the cation of PGH disrupted the plasma membrane via ionic interaction, as observed for PHMG-P. In addition, we suggest the disruption of plasma membrane as a molecular initiating event of cationic chemicals-induced adverse outcomes when exposed directly to the lungs.

Electronic supplementary material

The online version of this article (10.1007/s43188-020-00058-x) contains supplementary material, which is available to authorized users.

Introduction

Biocides are chemical substances or microorganisms that can kill, deter, render harmless, or control any harmful organism [1]. They have been used widely in healthcare products, cosmetics, household cleaning products, pet and general disinfectants, and food preservatives [1]. As public awareness of hygiene issues has increased, the use of biocides has also increased. Biocides help prevent the spread of infection and contamination of food or water, and prolong the shelf-life of various products [2, 3]. However, humans may be unintentionally exposed to them through ingestion or inhalation. Inhalation can occur owing to spraying or evaporation of biocide-containing products [4], which may cause health problems. The serious humidifier disinfectant-associated incidents that occurred in Korea in 2011 are a prominent example of this. To prevent the growth of microorganisms in a humidifier, humidifier disinfectants were widely used in Korean households [5, 6]; examples of such compounds are oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH), polyhexamethyleneguanidine phosphate (PHMG-P, poly(iminocarbonimidoylimino-1,6-hexanediyl) phosphate), and chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT). Particles of these biocides are generated by humidifiers, enter the human lungs, and cause severe respiratory disease [7].

PGH (CAS No. 374572-91-5) and PHMG-P (Cas No. 89697-78-9) are slightly toxic materials [8, 9]. Huntingdon Life Sciences Ltd. has conducted several toxicity tests of PHMG-P, including oral and dermal toxicity tests, skin and eye irritation tests, and the Ames test [9]. Generally, PHMG-P does not pose a public health concern when individuals are exposed to this chemical in formulated products [9] and it has been used widely in clinical and household products. However, PHMG-P is highly toxic when it is exposed directly to the lung. Some studies have shown the toxicities of PHMG-P after humidifier disinfectant-associated incidents [10–14], but few studies have assessed the toxicity of PGH. Buxbaum et al., reported that Akacid plus, a 3:1 mixture of PHMG and PGH, is an effective biocide with broad-spectrum antimicrobial properties and low toxicity. The oral and dermal LD₅₀ values of Akacid plus are above 2000 mg/kg for acute toxicity in rats [8]. However, there is no research report regarding the pulmonary toxicity of PGH.

An adverse outcome pathway (AOP) is a conceptual framework that organizes existing knowledge on linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment [15]. This term has been proposed to support the needs for assessing chemical safety of increasing number of chemicals to fulfill new legislative mandates, while minimizing reliance on resource-intensive testing approaches such as animal test [16]. AOP is composed of 3 key components; molecular initiating event (MIE), key event (KE), and key event relationships (KERs). MIE is the interaction of a chemical with a target biomolecule at the molecular level, for example, receptor/ligand binding and protein oxidation [17]. KE is a measureable biological change that is essential to the progression from an MIE toward a specific adverse outcome (AO) [16]. KER describes scientific relationship between a pair of KEs. AOP is not chemical specific. Chemicals that trigger a single MIE have a potential to produce a similar sequence of KEs [16]. Describing various KEs and KER linking an MIE and AO on a basis of data from a myriad of toxicity or mechanism studies provides systemic evidence of toxicity, with which we can extrapolate the potential hazard of new chemicals or elucidate new mode of action of the chemicals.

In this study, we aimed to investigate whether PGH can cause pulmonary toxicity following exposure through intratracheal instillation. We evaluated the single- and repeated-dose toxicity of PGH and PHMG-P following exposure through intratracheal instillation and compared the pathological progression induced by both chemicals. In addition, we explored the mechanism of PGH-induced cytotoxicity and compared the toxic mechanism of PGH with those of PHMG-P, which has already been elucidated in a previous study [19], and other chemicals.

Materials and methods

Chemicals

PGH (CAS No. 374572-91-5, 20.1% solution) was purchased from Shanghai Scunder Industrial Co., Ltd (Shanghai, China) and PHMG (Cas No. 89697-78-9, 25% solution) was kindly provided by SK Chemicals (Seongnam, Korea). Saline was purchased from Daihan Pharmaceutical Company (Ansan, Korea).

Animals

Seven-week-old male C57BL/6 mice with a body weight 17–21 g were purchased from Orient Bio Inc. (Seongnam, Korea). The mice were housed in environmentally controlled animal facilities, with the animal room maintained at 23 ± 3 °C, relative humidity of 50 ± 10%, air ventilation of 10–20 times/h, and light intensity of 150–300 lx, with 12 h light/dark cycle. Pelleted food for experimental animals (PM Nutrition International, Richmond, IN, USA) and UV-irradiated (Steritron SX-1; Daeyoung, Inc., Incheon, Korea), filtrated (1 μm) tap water were provided ad libitum. Mice were acclimatized for 9 days after arrival. All experiments were approved by the Institutional Animal Care and Use Committee of Korea Institute of Toxicology.

Experimental design

The experimental scheme is shown in Fig. 1a and b. For the single- and repeated-dose study, the doses of PGH and PHMG-P were set as 1.5 mg/kg and 0.6 mg/kg, respectively. In our previous study, 0.3 mg/kg, 0.9 mg/kg, and 1.5 mg/kg of PHMG-P were chosen as low, intermediate, and high doses [20]. The same doses of PGH were exposed to mice (Supplementary Figs. 2, 3 and Supplementary Table 1). Both PGH- and PHMG-P-treated mice showed lung inflammation, as revealed by histopathological examination. However, the inflammation resulting from PGH (0.3 mg/kg, 0.9 mg/kg, and 1.5 mg/kg) was relieved on day 14, whereas the inflammatory response induced by PHMG-P (0.3 mg/kg, 0.9 mg/kg, and 1.5 mg/kg) was exacerbated on day 14. To assess the time course of pathogenesis induced by each chemical, the dose of PGH was set as 1.5 mg/kg and that of PHMG-P was set as 0.6 mg/kg, as its inflammatory response was lower than that of PGH (Supplementary Figs. 2, 3 and Supplementary Table 1).

Fig. 1.

Experimental scheme and changes in body weights of mice instilled PGH or PHMG-P. Scheme of experimental design of the single- (a) and repeated-dose (b) study. Mice were intratracheally instilled once or three times with 1.5 mg/kg PGH or 0.6 mg/kg PHMG-P. The control group (control) was treated with saline through the same route. Body weights were measured at designated days. The numbers of mice for control, PGH, and PHMG-P group were 8, 10, and 10 for single-dose toxicity (c), and 15, 18, and 18 for repeated-dose toxicity (d), respectively. At each necropsy day, 4–6 mice per group were sacrificed. Red arrow indicates intratracheal instillation (I.T.I.)

For the single-dose study, mice were lightly anesthetized with isoflurane and 1.5 mg/kg PGH or 0.6 mg/kg PHMG in a volume of 50 μL was instilled intratracheally. The control group was administered saline via the same route. Body weight was observed on the day before instillation and three times per week after instillation. Mice were necropsied on days 8 and 15.

For the repeated-dose study, 1.5 mg/kg PGH or 0.6 mg/kg PHMG-P in a volume of 50 μL was instilled intratracheally to mice three times once every 4 days (days 1, 5, and 9). Our preliminary study showed that intratracheal instillation of saline twice per week did not adversely affect mice, that is, normal clinical signs occurred, or no pathological changes. To prevent the adverse effects of anesthesia as much as possible, instillation was performed once every 4 days. 1.5 mg/kg PHMG-P resulted in severe weight loss and lung injury; therefore, the frequency of treatment was set as three times, to ensure that the total dose did not significantly exceed 1.5 mg/kg. The control group was administered saline three times via the same route. Body weight was observed on the day before instillation and twice per week after instillation. To compare the time course in the pathogenesis induced by both chemicals, mice were killed on days 15 (6 days after the last instillation), 28 (19 days after the last instillation), and 36 (27 days after the last instillation) after the first instillation. At each necropsy day, mice were euthanized by isoflurane overdose, and the spleen, thymus, and lungs were removed and weighed. The left lungs, spleen, and thymus were then fixed in 10% neutral buffered formalin. The right lungs were snap-frozen for evaluation of gene and cytokine expression.

Cytokine measurement by ELISA

The frozen lungs were placed in phosphate buffered saline (PBS) containing 1% Triton X-100 at a ratio of 100 mg tissue per milliliter and then homogenized. The homogenates were incubated with shaking at 4 °C for 30 min and centrifuged at 13,000 rpm at 4 °C for 15 min. The levels of CXCL1, interleukin-1β (IL-1β), interleukin-6 (IL-6), and interferon-γ (IFN-γ) were quantified by using ELISA kits (R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s protocol. The total protein in the homogenates was determined by using the BCA protein assay (Sigma-Aldrich).

MCP-1 gene expression analysis

Total RNA was extracted from mouse lungs using RNeasy Mini kit (Qiagen). RNA yield and purity was measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, ED, USA). One microgram of the total RNA was reverse-transcribed to cDNA by using the Improm-II™ Reverse Transcription System (Promega, Madison, WI, USA). Monocyte chemoattractant protein-1 (MCP-1) mRNA levels were quantified using SYBR Green PCR Master Mix (Applied Biosystems, Woolston, Warrington, UK) on ABI StepOne (Applied Biosystems, Foster City, CA). β-actin was used as the internal control. The sequences of the primers were as follows: MCP-1 (Accession number: NM_011333.3) sense 5′-AGGTGTCCCAAAGAAGCTGTA-3′, antisense 5′-ATGTCTGGACCCATTCCTTCT-3′; β-actin sense (Accession number: NM_007393.5) 5′ CGTGCGTGACATCAAAGAGAA-3′, antisense, 5′-GGCCATCTCCTGCTCGAA-3′. Amplification efficiency of primer pairs was determined using relative standard curves. The real-time PCR cycle conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The relative gene expression was quantified using 2^−ΔΔCt method.

Histopathological examination

Lung, thymus, and spleen samples were fixed in 10% neutral formalin buffer for histological examination. From the fixed samples, 4-μm-thick paraffin sections were cut, stained with hematoxylin and eosin (H&E) and Masson’s trichrome (lungs only), and subjected to microscopic observation. The degree of lung inflammation and fibrosis was evaluated on a subjective scale by a pathologist. A scoring system is based on the presence or abundance of the following: chronic granulomatous inflammation/fibrosis (0, absent; 1, presence of lesions involving < 20% of the lung parenchyma; 2, lesions involving 20–40% of the lung; 3, lesions involving 40–60% of the lung; 4, lesions involving 60–80% of the lung; or 5, lesions involving > 80% of the lung), aggregate of macrophages (0, absent; 1, present in fewer than 10% of alveolar region; 2, present in more than 10% but less than 20% of alveolar region; or 3, present in > 20% of alveolar region), and infiltration of lymphocytes (0, absent; 1, the number of lymphocytic foci is present fewer than 5; 2, the number of lymphocytic foci is present more than 5 but less than 10; or 3, the number of lymphocytic foci is present more than 10).

Cell culture and cell viability assay

A549, human lung epithelial cells, WI-38, human lung fibroblasts, and THP-1, monocyte cells were obtained from Korean Cell Bank. A549 and THP-1 cells were suspended in RPMI 1640 medium supplemented with 10% FBS (Thermo Scientific) and penicillin/streptomycin. WI-38 cells were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin. THP-1 was differentiated into macrophage-like cells with 100 nM phorbol myristate acetate (PMA) (Sigma-Aldrich).

A549 and WI-38 cells were plated at a density of 5 × 10³ cells per well. THP-1 cells were seeded at a density of 1 × 10⁵ cells per well and differentiated with 100 nM PMA overnight. After 24 h, the cells were treated with 0.5–20 μg/mL PGH or PHMG-P. Cell viability was measured by using the cell counting kit (CCK)-8 (Dojindo, Japan) in accordance with the manufacturer’s protocol. Briefly, 10 μL CCK-8 was added to each well, and then incubated at 37 °C for 2 h. Optical density was measured at 450 nm by using a SpectraMax M3 microplate reader from Molecular Devices (Sunnyvale, CA, USA). Cell viability was expressed as a percentage of the vehicle control. All experiments were performed three to four times independently.

To evaluate the protective effect of DNA on PGH- or PHMG-P-induced cell death, A549 cells were plated at a density of 0.8 × 10⁴ cells per well by modifying a previously described method [23]. After 24 h, 12.5–100 μg/mL DNA and 20 μg/mL PHMG-P or PGH were added into each well. Cell viability was measured by the application of CCK-8 after 24 h of incubation, as described above.

Measurement of IL-1β production in THP-1 cells

THP-1 cells were seeded at 2 × 10⁶ cells per well and differentiated with 100 nM PMA overnight. After 24 h, the cells were washed with PBS three times and treated with PGH (15 μg/mL and 20 μg/mL) or PHMG-P (10 μg/mL and 20 μg/mL) for 24 h. To evaluate the protective effect of DNA, cells were cotreated with 12.5–50 μg/mL DNA and 20 μg/mL PGH or PHMG-P. After incubation for 24 h, the cell supernatant was collected and centrifuged at 13,000 rpm for 5 min. IL-1β was measured in cell supernatant in accordance with the manufacturer’s protocol (R&D Systems). All experiments were performed three times independently.

Measurement of reactive oxygen species (ROS) level in A549 cells

A549 cells were seeded at 0.8 × 10⁴ cells per well and incubated for 24 h. The cells were treated with PGH (10–30 μg/mL) or PHMG-P (5–20 μg/mL) with or without 3.13–25 μg/mL DNA for 6 h. After washing with PBS, 10 μM dichlorodihydrofluorescein diacetate (DCF-DA) was added to wells and incubated for 30 min. The fluorescence intensity was measured by using a SpectraMax M3 microplate reader (Molecular Devices) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescence intensity was adjusted to total cell viability detected by CCK-8, based on the method of Song et al. [22]. The experiment was performed three times independently.

Statistical analysis

One-way analysis of variance with Tukey’s or Dunnett’s T3 post hoc test was used to determine differences among groups (SPSS Ver15.0.0, SPSS Inc., USA). A p value of less than 0.05 was considered statistically significant. All data are presented as the mean ± standard deviation. The number of samples in each group is indicated in the figure legends.

Results

Body weight

The single-dose PGH (1.5 mg/kg) and PHMG-P (0.6 mg/kg) groups lost 4.9% and 9.6% of their body weights after instillation and subsequently started to gain weight (Fig. 1c). For the repeated-dose group, one mouse was found dead on day 12 or 2 mice were euthanized because the mice became moribund in the PHMG-P group. Mice in the PGH group showed approximately 8.4%, 9.9%, and 12.6% weight loss compared with weight of the control group on the next day after instillation but recovered rapidly from day 14 onward (Fig. 1d). Mice in the PHMG-P group showed approximately 8.6%, 17.1%, and 22.5% weight loss, respectively, and then slightly gained weight but did not recover to the control level until the end of experiment.

Oran weight

For the single-dose groups (Fig. 2a), lung weight in both groups increased significantly. Lung enlargement ameliorated on day 15 in the PGH group (51.3% on day 8 and 39.1% on day 15), whereas lung weight in the PHMG-P group increased more on day 15 (73.2%) than on day 8 (42.7%). Thymus weight of the PGH group increased by 31.3% on day 15). In contrast, thymus weight of the PHMG-P group decreased by 24.2% on day 8 but recovered on day 15. However, there was no change in relative weight (thymus weight/body weight) in either group (Supplementary Fig. 4).

Fig. 2.

Changes in organ weights of mice instilled PGH or PHMG-P. Mice were intratracheally instilled once (a) or three times (b) with 1.5 mg/kg PGH or 0.6 mg/kg PHMG-P. The control group was treated with saline through the same route. Organ weights of single- (a) and repeated-dose (b) groups were shown (n = 4–6). Data are expressed as mean ± SD; *p < 0.05, **p < 0.01 indicates significant difference from the control group

For the repeated-dose study (Fig. 2b), lung weight in both groups increased markedly. However, the pattern of increase in lung weight was different for each group. The increase in lung weight became slightly lower over time in the PGH group but was slightly higher over time in the PHMG-P group. Thymus weight slightly decreased by 17.2% on day 15 and increased by 19.3% on day 28 in the PGH group (Fig. 2b). In the PHMG-P group, thymus weight decreased on days 15, 28, and 36 by 55.5%, 50.2%, and 37.1%, respectively. No changes were observed in spleen weights in both groups.

Quantification of cytokines and MCP-1 gene expression in the lungs

For the single-dose groups (Fig. 3), IL-1β, IL-6, CXCL-1, and MCP-1 expression increased on day 8, but returned to the basal level on day 15 in the PGH group. IL-6 expression increased on days 8 and 15, but expression of the anti-fibrotic cytokine IFN-γ slightly decreased on day 15 in the PHMG-P.

Fig. 3.

Cytokine production in the lungs. The lung homogenates were used for quantification of CXCL-1, IL-1β, IL-6, and IFN-γ for single- (a) and repeated-dose (b) groups (n = 4–6). The mRNA expression of MCP-1 (c) was determined using real time PCR (n = 4–5). Data are expressed as mean ± SD; *p < 0.05, **p < 0.01 indicate significant difference from the control group. The quantity of IL-1β of repeated-dose PHMG-P group was beyond maximum limit in five of six animals at day 15 and three of six animals at day 36, so the maximum levels were applied

For the repeated-dose groups, the chemokine and cytokine levels increased overall. The PGH group showed increases in the expression of CXCL-1, IL-1β, and IL-6 on day 15, but the expression was subsequently decreased, with the exception of IL-1β. IL-1β and IL-6 levels increased markedly on days 15 and 28 in the PHMG-P group. MCP-1 gene expression also increased on days 28 and 36. In addition, the expression of IL-1β and IL-6 tended to decrease on day 28 but increased again on day 36. IFN-γ expression decreased in both groups on day 15, but was subsequently restored to normal.

Histopathological examination

Morphological changes in the lung, spleen, and thymus at each time point are presented in Fig. 4 and Supplementray Fig. 5. The degree of pathological changes in the lungs was summarized in Tables 1 and 2. Both PGH and PHMG-P induce immune cell infiltration and collagen deposition in lungs. In the single-dose PGH group, aggregation of foamy or alveolar macrophages in the alveoli, infiltration of lymphocytes in the perivascular and parenchymal regions, and minimal fibrosis were observed on day 8. These findings tended to return to normal on day 15. However, aggregation of foamy or alveolar macrophages and fibrotic changes were more severe in the single-dose PHMG group than in the PGH group, and these findings were not ameliorated on day 15.

Fig. 4.

Histopathological examination of PGH or PHMG-P–treated mice. Representative histological sections from lung tissues were shown. The lungs were stained with hematoxylin and eosin (H & E) (a, c) and Masson’s trichrome (b, d). Single- (a, b) and repeated-dose (c, d) lung sections were shown. Blue arrow indicates foamy/alveolar macrophage infiltration, red arrow indicates lymphocyte infiltration, black arrow indicates degeneration of bronchiolar epithelium, black triangle indicates fibrosis, green arrow indicates cellular debris, and orange triangle indicates bronchoalveolar hyperplasia. Scale bar represents 50 μm (original magnification, ×200)

Table 1.

Quantitative histopathological evaluation of the lung tissues in the single-dose PGH and PHMG-P groups

	Control	PGH	PHMG-P
Aggregate of macrophages
Day 8	0	1.20 ± 0.45**	1.60 ± 0.55**
Day 15	0	0.80 ± 0.45*	2.40 ± 0.89**
Infiltration of lymphocytes
Day 8	0	2.80 ± 0.45**	2.40 ± 0.55**
Day 15	0	1.20 ± 0.45**	1.60 ± 0.55**
Chronic granulomatous inflammation/fibrosis
Day 8	0	0.60 ± 0.55	1.80 ± 0.45**
Day 15	0	0	1.80 ± 0.84

Data represents mean ± SD. *p < 0.05, **p < 0.01

Table 2.

Quantitative histopathological evaluation of the lung tissues in the repeated-dose PGH and PHMG-P groups

	Control	PGH	PHMG-P
Aggregate of macrophages
Day 13	0	2.00 ± 0.00**	2.20 ± 0.45**
Day 28	0	1.17 ± 0.41**	2.50 ± 0.55**
Day 36	0	1.00 ± 0.63	2.33 ± 0.82**
Infiltration of lymphocytes
Day 13	0	1.50 ± 0.55	2.40 ± 0.55**
Day 28	0	2.00 ± 0.63**	2.00 ± 0.00**
Day 36	0	1.83 ± 0.41**	1.67 ± 0.52**
Chronic granulomatous inflammation/fibrosis
Day 13	0	1.33 ± 0.52**	3.20 ± 0.84**
Day 28	0	1.00 ± 0.00	3.67 ± 0.52**
Day 36	0	1.00 ± 0.63*	3.17 ± 0.75**

Data represents mean ± SD. *p < 0.05, **p < 0.01

In the repeated-dose PGH group, the aggregation of foamy or alveolar macrophages and pulmonary fibrosis tended to be similar or become slightly restored over time. However, infiltration of lymphocytes into the lungs slightly increased over time. Cellular debris in the alveoli was observed on days 28 and 36. In the repeated-dose PHMG-P group, aggregation of foamy or alveolar macrophages and pulmonary fibrosis slightly increased with time, whereas lymphocyte infiltration in the perivascular and parenchymal region slightly decreased over time. In addition, bronchiolar–alveolar hyperplasia and degeneration/regeneration of the bronchiolar epithelium were observed. These pathological changes were exacerbated over time.

In the single-dose PGH and PHMG-P groups, there was no histopathologic change in the spleen and thymus (Supplementary Fig. 5). In the repeated-dose PGH and PHMG-P groups, extramedullary hemopoiesis was observed in both groups. This change was fully restored from day 28 in the PGH group, but was exacerbated over time in the PHMG-P group (Supplementary Fig. 5B). Minimal thymic atrophy was observed only in the PHMG-P group and was sustained until the end of the experiment (Supplementary Fig. 5D).

Cell viability and protective effect of DNA on PGH or PHMG-P-induced cytotoxicity

To evaluate the toxic effects of PGH and PHMG-P, cell viability was assayed by using CCK-8. The viability of A549, THP-1, and WI-38 cells were decreased by PGH and PHMG-P in a dose-dependent manner (Fig. 5a–c). Treatment with 10 μg/mL PGH or PHMG-P resulted in 49.2% and 20.7% viability in A549 cells, 95.4% and 77.0% viability in THP-1 cells, and 70.3% and 41.6% viability in WI-38 cells, respectively. Generally, PHMG-P was more cytotoxic than PGH to all cell lines.

Fig. 5.

Cell viability of A549, THP-1 and WI-38 exposed to PGH or PHMG-P. a–c A549, THP-1, and WI-38 cells were incubated with 0.5–20 μg/mL PGH or PHMG-P for 24 h. d, e A549 cells were coincubated with 12.5–100 μg/mL DNA and 20 μg/mL PGH or PHMG-P for 24 h. Cell viability was measured by using CCK-8 and expressed as a percentage of the vehicle control. Data are expressed as mean ± SD of three to four separate experiments; *p < 0.05, **p < 0.01 indicates significant difference from the vehicle control

To clarify whether PGH induces cytotoxicity through its cationic nature, anionic DNA was added concomitantly with PGH. The addition of DNA in the presence of PGH or PHMG-P increased cell viability in a DNA dose-dependent manner (Fig. 5d, e).

Protective effect of DNA on PGH or PHMG-P-induced IL-1β and ROS production

IL-1β and ROS production was measured in PMA-differentiated THP-1 cells and A549 cells, respectively. Both chemicals increased IL-1β secretion and ROS production in a dose-dependent manner (Fig. 6). In particular, PHMG-P induced 2.2–3.5 times more ROS than that by PGH at the same concentration (10 μg/mL and 20 μg/mL).

Fig. 6.

Protective effect of DNA on PGh or PHMG-P induced IL-1β and ROS production. a–d IL-1β production was measured using THP-1 cells. PGH (15 μg/mL and 20 μg/mL) or PHMG-P (10 μg/mL and 20 μg/mL) was incubated with or without 12.5–50 μg/mL DNA for 24 h. Cell supernatants were used for measurement of IL-1β. e–h For ROS detection, A549 cells were incubated with PGH (10–30 μg/mL) or PHMG-P (5–20 μg/mL) with or without 3.13–12.5 μg/mL DNA for 6 h. Cells were washed with PBS and DCF-DA was added to each well. After 30 min, fluorescence intensity was measured. Data are expressed as mean ± SD; *p < 0.05, **p < 0.01 indicate significant difference from the vehicle control. ^#p < 0.05, ^##p < 0.01 indicate significant difference from PGH or PHMG-P alone

Next, to evaluate the protective effect of DNA on PGH- or PHMG-P-induced cytokine and ROS production, cells were cotreated with DNA and PGH or PHMG-P. Cotreatment of DNA with PGH or PHMG-P decreased IL-1β and ROS production in a DNA dose-dependent manner compared with that after treatment of PGH or PHMG-P alone.

Discussion

Approximately 90 workers who were exposed to high concentrations of aerosolized paint at textile-printing factories suffered from severe respiratory disease, and 6 workers of them died in Spain in 1992. This was called as the “Ardystil syndrome”. A similar outbreak took place in Algeria, where one woman died and at least two workers were affected by severe interstitial lung disease [23, 24]. The causative chemicals, Acramin FWR, Acramin FWN, and Acrafix FHN, resulted in pulmonary inflammation and fibrosis when given intratracheally in hamsters. However, these chemicals are less toxic when administered by oral route in hamsters. The oral LD₅₀ of these chemicals are above 2000 mg/kg [25]. Hoet et al., showed that these chemicals exert cytotoxicity by virtue of the polycationic character in their multiple nitrogen atoms. The cytotoxicity induced by Acramin FWR, Acramin FWN, and Acrafix FHN was significantly decreased in the presence of polyanionic compounds such as sulodexide, DNA, or poly-l-glutamic acids [21]. Like Acramin FWR, Acramin FWN, and Acrafix FHN, PHMG-P is less toxic when given by oral or dermal routes [9]. However, they caused interstitial lung disease in humans and rodents when directly exposed to the lungs. From these results, Hoet et al. [26] proposed that these chemicals caused pulmonary toxicity through the same mechanism; namely, their polycationic nature. In this study, we evaluated the toxic mechanism of PGH, another polycationic compound. Until now, few studies have assessed the inhalation toxicity of PGH. Therefore, we assessed the pulmonary toxicity of PGH and compared the toxicity of PGH with that of PHMG-P.

Exposure of the lungs to both PGH and PHMG-P caused an inflammatory response and fibrotic changes in the lungs. Immune cells such as alveolar macrophages, neutrophils, and lymphocytes infiltrated in alveoli, perivascular and parenchymal region and proinflammatory cytokines and chemokines also upregulated. In addition, collagen deposition was observed in the lung parenchyma. However, the pathological progression caused by both chemicals was somewhat different. In the single- and repeated-dose study, elevated proinflammatory cytokine levels at early stage decreased over time in the PGH group, whereas the upregulated cytokine level was slightly decreased or remained at the same level over time in the PHMG-P group. Moreover, bronchoalveolar hyperplasia, degeneration of the bronchiolar epithelium, and pulmonary fibrosis were exacerbated in PHMG-P groups, however, these pathological changes ameliorated over time in the PGH group. In our previous study, we showed that even the group with 0.3 mg/kg PHMG-P, which exhibited no body weight loss and low cytokine production on day 7, showed more severe lung injury on day 14 [20]. However, the PGH group showed lower inflammatory responses on day 14, although the grade of inflammation in the PGH group was higher than that in the 0.3 mg/kg PHMG group on day 7 (Supplementary Table 1). In addition, the high grade of inflammation did not induce pulmonary fibrosis in the PGH group (Supplementary Table 1). Overall, the inflammatory response appeared to be gradually resolving in the PGH group but seemed to be progressing in the PHMG-P group.

To restore tissue homeostasis following damage, inflammation should be appropriately resolved, and the repair process must be tightly regulated [27]. During the resolution of inflammation, infiltrating immune cells are removed by apoptosis or exodus from the inflamed site through draining lymphatics, and anti-inflammatory and pro-resolving mediators are produced [27–29]. Macrophages are abundant in immune cells after lung injury, as alveolar macrophages are resident in the lung, and circulating monocytes are recruited at the inflamed site and differentiate into macrophages. Macrophages can release a wide variety of compounds, including cytokines, chemokines, proteolytic enzymes, and other inflammatory mediators, and play an important role in inflammatory response and repair process by phenotype switching [30, 31]. The cytokine IL-1β is mainly secreted by monocytes and macrophages. It is a potent proinflammatory and profibrotic cytokine [32]. The accumulation of macrophages and sustained IL-1β production are detected in persistent inflammation [31, 33, 34]. Persistent monocyte influx and elevated MCP-1 levels are positively correlated with the severity of lung failure in patients with acute respiratory distress syndrome [35]. Thepen et al. [36] reported that local depletion of activated macrophages resulted in the disappearance of other immune cells and resolution of inflammation in a sodium lauryl sulfate-induced model of cutaneous inflammation. These studies indicated that elimination of activated macrophages is crucial for the resolution of acute inflammation. Sustained high number of infiltrated macrophages and elevated levels of IL-1β and MCP-1 in the lung tissues of the PHMG-P group may be the cause of the more severe pathological changes than those observed in the PGH group, although the phenotype of infiltrated macrophages requires further investigation.

PGH and PHMG induced cytotoxicity in a dose-dependent manner. Both chemicals produced the proinflammatory cytokine IL-1β and ROS, which can induce cell death and tissue damage. Interestingly, PHMG-P resulted in larger quantities of ROS than PGH at the same concentration, which contributes to the higher toxicity of PHMG-P. ROS help the host in removal of the microorgaisms but also damage cell and tissue function by oxidizing lipids and DNA and disturbing the structure of proteins [37]. The lung is vulnerable to oxidative injury because it has a vast surface area owing to the airway epithelium, alveolar ducts, and capillaries that are directly in contact with oxygen from gas exchange between the air and blood [38]. Increased ROS levels generated by chemical stimuli initiate lung inflammatory responses by activation of NF-κB, which regulates the gene expression of proinflammatory mediators [39, 40]. Generation of large quantities of ROS by PHMG-P may contribute to prolonged and chronic lung injury.

PGH and PHMG are polymers containing a guanidine group (Fig. 7). Guanidine is in a protonated state in physiological condition [41]. The positive charges in these cationic biocides interact with bacterial membranes owing to their opposite charge, causing the leakage of cellular components and cell death [18]. In our previous study, we showed that PHMG-P induces cytotoxicity by disrupting the cellular membrane owing to the cationic nature of the guanidine group [19]. Neutralization of the cationic charge of PHMG-P with the anion of DNA restored the viability of various human cell lines and increased the survival of zebrafish embryos [19]. In the present study, we showed that PGH induced cell death through the same mechanism as that of PHMG-P. Cotreatment with DNA and PGH increased cell viability and diminished cytokine and ROS production in a DNA dose-dependent manner. Hoet et al. [21] also showed that nitrogen-containing polymers such as Acramin FWR, Acramin FWN, and Acrafix FHN, exert cytotoxicity through the same mechanism of action as those of PGH and PHMG-P. Taken together, it is reasonable to predict that polycationic chemicals are highly toxic when exposed to the lungs, although they have low toxicity when administered via other routes than inhalation. In addition, we suggest that the disruption of cellular membrane by ionic interaction between cellular membrane and these chemicals is an MIE for the adverse outcomes induced by these polycationic chemicals. To validate if the membrane disruption is an MIE for the cationic chemicals-induced AO (lung inflammation and fibrosis), more scientific evidences must be accumulated in the future.

Fig. 7.

Chemical structures of PGH (a) and PHMG-P (b). PGH and PHMG-P have a guanidine group. The chemical formula of guanidine is HN = C(NH₂)₂

Although both PGH and PHMG-P cause cytotoxicity through cationic nature and cause cytokine production and collagen deposition in the lungs, the degree of toxicity or pathogenesis of each chemical is different. It appeared to be dependent on the chemical structure and charge density of these cationic polymers. As the plasma membrane is flexible, it can easily be deformed upon interaction with cationic polymers. Polymers can be inserted into the plasma membrane or adsorbed onto the plasma membrane surface, depending on the chemical structure of the polymers [18, 42]. Recently, Shim et al. [43] showed that over 70% of inhaled or intratracheally instilled PHMG-P is retained in the lungs for 7 days. It may be possible that PHMG-P is integrated into or tightly adsorbed on the plama membranes or organelle membranes; therefore, it is not readily cleared from the lungs and may induce persistent inflammation and fibrogenesis in the lungs. Considering our data, the behavior of PGH in the lungs appeared to be different from that of PHMG-P. In the single- and repeated-dose study, the PGH group showed resolution of pulmonary inflammation with minimal fibrotic changes over time, which may have resulted from the rapid clearance of PGH from the lungs. Further studies are required to investigate the clearance of PGH from the lung (Fig. 3).

We showed that PGH induced pulmonary inflammation and fibrotic changes when directly exposed to the lungs and that inhaled PHMG-P was more toxic than inhaled PGH, with 1.5 mg/kg PGH showing lower body weight loss, smaller changes in organ weight, and lower grade of inflammatory and fibrotic changes than those induced by 0.6 mg/kg PHMG-P. These results were consistent with the cell viability data in various cell lines. In addition, we confirmed that the action mechanism of PGH was similar to that of PHMG-P, whereby it disrupted the plasma membrane through ionic interaction. We suggested that the disruption of plasma membrane is an MIE of lung inflammation and fibrosis induced by polycationic chemicals.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.