Short-term toxicity of dibutyl phthalate to mice intestinal tissue

Abstract

The objective of this study was to investigate changes in intestinal histopathology and expression of heat-shock proteins (HSPs) in the small intestinal tissue of mouse after acute exposure to dibutyl phthalate (DBP). Forty-eight 60-day-old Institute of Cancer Research (ICR) mice were administered DBP by gavage once a day for 10 days. The mice were divided into three groups of 16 mice each: the high-dose group was administered 500 mg/kg body weight (BW) DBP; the low-dose group was administered 50 mg/kg BW; and the control group was not administered DBP. Significant increases in the uterine index, ovary index, and testicular index were observed in the DBP-exposed groups compared to those in the control group. Villus height and V/C ratio significantly increased (p < 0.05) in the duodenum and decreased (p < 0.05) in the jejunum after the administration of DBP. The goblet cell number decreased in both the duodenum and the jejunum of mice exposed to DBP (p < 0.05) compared to the number in the control group mice. Damage to the structure of the small intestine was accompanied by a marked increase in HSP27 expression and a decrease in the expression of HSP70 and HSP90 in both high-dose and low-dose groups. These results indicate that elevated HSP27 levels in the duodenum and jejunum may be important markers for acute DBP exposure and that HSP27 may act as a protective protein involved in intestinal mucosa repair.

Keywords

Dibutyl phthalate intestinal tissues heat-shock proteins acute toxicity

Introduction

Dibutyl phthalate (di-n-butyl phthalate; DBP) is widely used as a plasticizer in a variety of household products, including food packaging materials, infant products, cosmetic products, toys, food containers, furniture, cosmetics, personal care products, latex adhesives, cellulose plastics, varnish, and dye solvents (Duty et al., 2005; Hauser et al., 2007; Shea, 2003; Stales et al., 1997; Wilkinson and Lamb, 1999). As a plasticizer, DBP does not polymerize with the polymer carbon chain during the manufacture of plastic and plastic products. Therefore, DBP is gradually released and enters the environment and has the potential to cause harm to the environment and living organisms (Fujii et al., 2003). Studies have shown that the concentration of DBP is high in the air (Rudel et al., 2003). However, DBP in the air or adhered to dust can be degraded by various types of bacteria within a few days (Cheung et al., 2007; Hara et al., 2010; Whangsuk et al., 2015; Wu et al., 2012). DBP in the soils, which cannot be degraded by bacteria, can enter the groundwater, thereby polluting the water source. If absorbed by crops, it can be indirectly transferred to humans or livestock, thus entering the food chain (Wang et al., 2015). A recent report showed that the toxic and apoptotic effects of DBP in mouse neocortical neurons in primary cultures were mediated via the aryl hydrocarbon receptor (Wójtowicz et al., 2017).

The intestinal tract is an important component of the human digestive system and immune system and contains large numbers of lymphocytes (Mowat and Agace, 2014). Therefore, as the largest immune organ in the human body, the intestine plays an important role in human health. Xu et al. (2015) have shown that DBP in aquatic environment can inhibit the neutrophils and macrophage formation in a concentration-dependent manner and have greatly influence on the immune system in fish.

Heat-shock proteins (HSPs) are known to function as molecular chaperones during protein assembly (Haslbeck et al., 2005), protein folding and unfolding (Zietkiewicz et al., 2004), and refolding of damaged proteins (Marques et al., 2006) for the protection and repair of cells and tissues. Studies have shown that the induction of HSP expression in intestinal epithelial cells is an important defense mechanism that protects the gut epithelium against a variety of stress factors, including oxidative stress, inflammation, bacterial infection, and hypoxia/reoxygenation injury (Arnal and Lallès, 2016; Liu et al., 2014a; Yuan et al., 2010).

Many studies have focused on the reproductive toxicity of DBP, but little is known about the effects of DBP toxicity on the gut. It is also not known whether HSPs play a protective role in the intestine after acute DBP exposure. Therefore, the present study was designed to investigate the effects of DBP on intestinal morphology and histology and the expression of HSPs in the intestinal tract of mice acutely exposed to DBP.

Materials and methods

Animals

Forty-eight 60-day-old ICR mice (24 females and 24 males) weighing about 20 g were provided by the Experimental Animal Center of Zhejiang Wanli University (Ningbo city, China). The animals were maintained in 12 individual cages (four mice per cage), housed in an air-conditioned animal room at 22 ± 3°C and 55 ± 10% humidity with a 12/12-h light–dark cycle, and allowed free access to a commercial feed and water ad libitum. Mice were acclimatized for 1 week before being used for subsequent experiments.

Treatment

DBP (Sigma-Aldrich, Shanghai, China) was dissolved in corn oil (99.5% pure, Shanghai Solvent Factory, Shanghai, China). The dosage levels and days of administration were determined based on the results of a previous study in which DBP was administered by gastric intubation at 500 mg/kg body weight (BW; high-dose group, 16 mice per group) and 50 mg/kg BW (low-dose group, 16 mice per group); the control group (16 mice per group) was not administered DBP. The volumes of DBP solution of the high-dose group and the low-dose group were calculated according to mice weight, and then the volumes were adjusted to 1 ml using corn oil before gavage. The animals of the control group were given 1 ml of corn oil by gavage. DBP was administered once a day for 10 days. At the end of the 10-day treatment period, all mice were euthanatized under ether anesthesia. Tissues samples (duodenum and jejunum) for histological analysis and immunohistochemical analyses were fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. All animal experiments were conducted in accordance with China legislation for the use and care of laboratory animals and were approved by Zhejiang Wanli University.

Histological analysis

After 24 h of fixation, the duodenum and jejunum samples were dehydrated in a graded series of ethanol and embedded in a paraffin block. Sections were serially sliced at a thickness of 5 µm, mounted on polylysine-coated slides, and stained with hematoxylin and eosin (HE). The morphological structure of the duodenum and jejunum, including intestinal villus height, crypt depth, intestinal muscle layer thickness, and goblet cell number, was measured using a Nikon 80i Microscope (Nikon, Tokyo, Japan) with a Biological Color Microscopy Image Analysis System (Aliakbarpour et al., 2012). The villus height was measured from the gland villus junction to the top of the villus. Ten of the longest intestinal villi were randomly selected from each slice, and the average length of the 10 villi was calculated for subsequent analyses. The crypt depth was measured from the gland villus junction to the base of the gland. The number of goblet cells was counted in five of the longest intestinal villi and expressed as a percentage of total epithelial cells (i.e. number of goblet cells per 100 epithelial cells). The cell count plate was used to define a reference length before measurement.

Immunohistochemical analysis

The paraffin-embedded, 5-µm-thick, serial tissue sections were deparaffinized and subsequently passed through decreasing concentrations of ethanol into water. Endogenous peroxidase activity was quenched with 3% (v/v) hydrogen peroxide in methanol for 15 min at room temperature (RT). The sections were placed in 0.01 M citric acid buffer (pH 6.0) and then heated in a microwave oven at 800 W for 3 min followed by 400 W for 10 min. Nonspecific antibody binding was blocked by incubating with normal bovine serum albumin (BSA; diluted and ready to use, AR1006, Wuhan Boster Biological Technology, China) for 20 min at RT. The sections were then incubated overnight at 4°C in a humidified chamber with a primary antibody diluted in Tween 20/Tris-buffered saline containing 0.5% BSA. The HSP27-specific monoclonal antibody (rabbit anti-HSP27 monoclonal antibody, ab49919, Abcam, UK) was used at 1:100 dilution, HSP70-specific monoclonal antibody (rabbit anti-HSP70 monoclonal antibody, ab74072, Abcam) was used at 1:200 dilution, and HSP90-specific monoclonal antibody (rabbit anti-HSP90 monoclonal antibody, ab13494, Abcam) was used at 1:200 dilution. To detect bound antibodies, the sections were incubated with a biotinylated secondary antibody (diluted and ready to use, BA1081, Wuhan Boster Biological Technology) for 20 min at 37°C and then incubated with an avidin-biotin-horseradish peroxidase system (diluted and ready to use, SA1022, Wuhan Boster Biological Technology) for 20 min at 37°C; this was followed by visualization with the diaminobenzidine substrate (diluted and ready to use, AR1025, Wuhan Boster Biological Technology). After each incubation step, the sections were washed in PBS (pH 7.4). In the control group, the primary antibody was replaced with blocking serum. Nuclear counterstaining was performed using hematoxylin. Finally, the tissue sections were dehydrated, mounted, and photographed. Relative levels of immunostaining for the three HSPs in the duodenum and jejunum were quantified by determining the intensity of the optical region of each section using Image Pro-Plus Software (Image Pro-Plus 6.0, Media Cybernetics, MD, USA). Briefly, 10 digital images at 1000× magnifications were captured by the Nikon 80i Microscope (Nikon, Tokyo, Japan). The measurement parameters included area sum and integrated optical density (IOD) sum. The optical density was calibrated and the area of interest was set through: hue: 0–30, saturation: 0–255, and intensity: 0–200; then images were converted to grayscale images, and the values were counted. The average optical density was based on the following formula: density mean = IOD sum/area sum (Wang et al., 2009; Xavier et al., 2005).

Statistical methods

Statistical analysis of differences between experiment groups and the control group was carried out using one-way analysis of variance with the Statistical Package for Social Sciences (SPSS; version 18.0). The mean values of each group were compared using Duncan’s test for multiple comparisons. Differences were regarded as significant and highly significant at p < 0.05 and p < 0.01, respectively.

Results

Changes in BW and organ indexes after DBP exposure

As shown in Table 1, the ovary (p < 0.01) and testis indexes (p < 0.01) of animals treated with DBP at doses of 50 and 500 mg/kg BW increased significantly compared to those of the control animals. Uterine indexes significantly increased in both low-dose (p < 0.05) and high-dose (p < 0.01) groups compared to those of the control group. Although we observed some minor changes in the heart, liver, and kidney indexes, as well as in BW in response to DBP exposure, the values were not significantly different from those of the control animals.

Table 1.

Changes in body weight and organs indexes after dibutyl phthalate exposure in mice.

	Heart (mg/g)	Liver (mg/g)	Kidney (mg/g)	Ovary^a (mg/g)	Uterine^a (mg/g)	Testis^a (mg/g)	Body weight (g)
Control	3.17 ± 0.21	37.12 ± 3.12	7.94 ± 0.71	0.14 ± 0.01B	3.97 ± 0.52B	2.21 ± 0.20B	25.2 ± 2.1
Low-dose group	3.03 ± 0.25	30.80 ± 2.96	8.04 ± 0.79	0.27 ± 0.02A	4.95 ± 0.42B	3.25 ± 0.28A	26.4 ± 2.3
High-dose group	3.19 ± 0.53	34.31 ± 3.23	9.16 ± 0.85	0.30 ± 0.02A	6.03 ± 0.56A	4.76 ± 0.41A	27.0 ± 2.3

^a Different uppercase letters in the same row denote significance at the 0.01 level, respectively. Data are expressed as means ± SD (n = 16).

Changes in intestinal morphology after DBP exposure

Figure 1 showed the morphology of the duodenum and jejunum in mice exposure to DBP. The duodenum and jejunum mucosa structures were intact in the control group (Figure 1(a) and (d)). The intestinal villus and crypt of the duodenum and jejunum showed some variation among mice in the experimental groups (Figure 1(b), (c), (e), and (f)). Changes in intestinal villus height, crypt depth, and the villus height/crypt depth ratio (V/C ratio) after DBP exposure were observed in Figure 2. The administration of 500 mg/kg BW DBP resulted in significant increases (p < 0.05) in villus height in the duodenum compared to that in the control group (Figure 2(a)). As for the jejunum, the intestinal villus height was significantly reduced (p < 0.05) in both low-dose and high-dose groups compared to that in the control group (Figure 2(d)). Crypt depth in the duodenum was lower in both low-dose (p < 0.05) and high-dose (p < 0.05) groups (Figure 2(b)) than in the control group. As for the crypt depth in the jejunum, there were no significant differences among the three groups (p > 0.05; Figure 2(e)). DBP administration markedly elevated the V/C ratios in high-dose (p < 0.05) group compared to those of the control group (Figure 2(c)). Conversely, the V/C ratio decreased in the jejunum in both low-dose (p < 0.05) and high-dose (p < 0.05) groups compared to that in the control group (Figure 2(f)).

The number of goblet cells in every 100 columnar epithelial cells of the small intestine was counted (Figure 3). With increasing DBP concentration, the percentage of goblet cells in the duodenum (Figure 3(a)) and jejunum (Figure 3(b)) in both low-dose and high-dose groups was significantly lower than that in the control group (p < 0.05).

Changes in HSP27 expression in intestinal tissues after DBP exposure

The effects of DBP on the expression of HSP27 in the duodenum and jejunum are shown in Figure 4. HSP27-positive signals were mainly distributed in the cytoplasm of villous epithelial cells in the duodenum and jejunum of all experimental groups. Interestingly, HSP27 expression in the cytoplasm of villous epithelial cells was more prominent in intact areas than in degenerated areas (Figure 4(b), (c), and (e)). Analysis of the relative levels of immunostaining among the groups showed that the expression of HSP27 in the duodenum and jejunum of both low-dose and high-dose groups was significantly higher (p < 0.01) than that in the control group. The levels of HSP27 increased sharply in the jejunum and reached a peak level (4.2-fold increase compared with control) in the low-dose group (Table 2).

Table 2.

Relative levels of immunostaining of three HSPs in duodenum and jejunum after dibutyl phthalate exposure.^a

	HSP27		HSP70		HSP90
	Duodenum	Jejunum	Duodenum	Jejunum	Duodenum	Jejunum
Control	0.0241 ± 0.0089B	0.0513 ± 0.0010C	0.1947 ± 0.0560A	0.1667 ± 0.0057Aa	0.1131 ± 0.0299A	0.1286 ± 0.0070Aa
Low-dose group	0.0995 ± 0.0201A	0.2147 ± 0.0275A	0.1393 ± 0.0087B	0.1001 ± 0.0011Bc	0.0036 ± 0.0008B	0.0452 ± 0.0114Bc
High-dose group	0.1019 ± 0.0134A	0.1279 ± 0.0027B	0.0940 ± 0.0054B	0.1141 ± 0.0078Bb	0.0977 ± 0.0053A	0.0670 ± 0.0059Bb

HSP: heat-shock protein.

^a Different uppercase and lowercase letters in the same row denote significance at the 0.01 and 0.05 levels, respectively. Data are expressed as means ± SD (n = 16).

Changes in HSP70 expression in intestinal tissues after DBP exposure

The effects of DBP on the expression of HSP70 in the duodenum and jejunum are shown in Figure 5. No obvious differences in HSP70 distribution in the duodenum and jejunum of the DBP-exposed groups and the control group were observed. HSP70-positive signals were mainly distributed in the cytoplasm of villous epithelial cells in the duodenum and jejunum of all experimental groups. Analysis of the relative levels of immunostaining showed that the expression of HSP70 in both low-dose and high-dose groups was significantly lower (p < 0.01) than that in the control group (Table 2).

Changes in HSP90 expression in intestinal tissues after DBP exposure

The effects of DBP on the expression of HSP90 in the duodenum and jejunum are shown in Figure 6. Similar to those of the other HSPs analyzed, HSP90-positive signals were mainly distributed in the cytoplasm of villous epithelial cells in both the duodenum and jejunum. Analysis of the relative levels of immunostaining showed that the expression of HSP90 in the duodenum of the low-dose group was significantly lower (p < 0.01) than that in the control group. However, there were no significant differences in the expression of HSP90 in the duodenum between the high-dose group and the control group. The relative levels of HSP90 in the jejunum of both low-dose and high-dose groups were significantly lower (p < 0.01) than that in the control group (Table 2).

Discussions

DBP is widely used in food additives, household products, and food packaging (Cartwright et al., 2000). The market demand for DBP has been increasing with the process of industrialization. Although DBP can be metabolized and excreted from the body, a proportion that cannot be metabolized will gradually accumulate and may have differing degrees of influence on the body (Jin et al., 2012; Meng et al., 2015; Prasad and Suresh, 2015).

In the present study, we showed that DBP exposure resulted in a significant increase in uterine, ovarian, and testicular indexes in both low-dose and high-dose groups, indicating that short-term DBP exposure can promote the development of reproductive organs in 8-week-old mice. DBP has been shown to cause reproductive endocrine disruption in various animal models, including increased estradiol and follicle-stimulating hormone levels (Chang et al., 2007). The study by Zhou et al. (2017) showed that exposure to the phthalate mixture increased uterine weight both in the F2 and F3 generations. It is possible that the DBP-induced increase in uterine weight may be the result of disrupted hormone levels or disrupted uterine development in adult mice. It was also confirmed that DBP administered to rats during the second half of pregnancy caused adverse effects on the reproductive development in male fetuses (Ema et al., 1998). The increase of testicular index in the present investigation reflects its susceptibility to DBP with the potential to interfere with critical reproductive function of testis. Histological studies by Nair (2015) indicated a dose-related degeneration of germinal, Leydig and Sertoli cells along with the loss of spermatozoa in the lumen.

This article also investigated whether different concentrations of DBP have toxic effects on the intestinal tissues of mice. We observed that the administration of DBP at a high-dose (500 mg/kg) markedly increased the villus height and the V/C ratio in the duodenum. However, villus height and the V/C ratio in the jejunum decreased significantly after exposure to the high concentration of DBP, indicating that intestinal morphology was altered at varying degrees after intragastric administration of DBP. A previous study showed that the intestinal tract plays an important role in digestion and absorption of nutrients, and the length and the width of the intestinal villi are positively correlated with the ability to absorb nutrients (Chwen et al., 2013). Crypt depth can mainly reflect the rate of epithelial cell formation. The epithelial cells migrate from the base of the crypt to the end of the villi to form an absorptive villous cell to complement normal shedding (Marshman et al., 2001; Wongdee et al., 2013). Therefore, long villi, short crypts, and a high V/C ratio reflect a healthy digestive system with high brush border enzyme activity (Wang et al., 2013). The significant reduction in the villus height and the V/C ratio in the duodenum after exposure to the high concentration of DBP indicated that DBP has an adverse effect on the absorptive capacity of the jejunum. Ozaki et al. (2017) examined the hydrolytic metabolism of DBP using rat tissue microsomes and found that small intestinal microsomes exhibit higher activity toward long-side-chain phthalates. Therefore, the present findings that DBP promotes the growth of villi in the duodenum and has a damaging effect on the absorptive capacity of the jejunum suggest that DBP can cause tissue-specific effects in the intestine. We speculate that the possible reasons for the different effects of DBP on the duodenum and jejunum are that the duodenum may be able to metabolize DBP or that the jejunum may over-absorb DBP. However, further research is required to investigate these hypotheses.

Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion (Kober et al., 2014). The results of the present study showed that the number of goblet cells in the duodenum and jejunum of the low-dose group and the high-dose group decreased significantly after DBP exposure. Numerous studies have indicated that the goblet cells in the intestine play a key role in the reconstitution of intestinal mucosa after hemorrhagic shock, inflammation, and infection, or chemotherapy-induced injury (Hino et al., 2015; Pelaseyed et al., 2014; Zuo et al., 2015). The decrease in goblet cell number may reduce mucus secretion, thereby reducing the functional capacity of the mucus barrier and increasing the likelihood of detrimental tissue invasion.

Furthermore, we also evaluated the expression of HSPs in intestinal epithelial cells after DBP exposure. We concluded that the elevation in HSP27 levels occurs in the duodenum and jejunum of ICR mice 10 days after DBP exposure. This result indicates that HSP27 plays an important role in the local immune system in the gut and is in agreement with the fact that HSPs have multiple functions related to intestinal health and disease (Liu et al., 2012). Many studies have shown that HSP27 plays an important role in maintaining cell structure (Henderson and Pockley, 2010; Tashiro et al., 2001), and HSP27-mediated stabilization of microfilaments is thought to be responsible for the increased survival of cells recovering from cellular stress. Our immunohistochemical study results also revealed that HSP27-positive staining was distinctly lower in the cytoplasm of granular degenerated areas, indicating a declining protective effect of HSP27 on mucous epithelial cells.

The expression of HSP70 can be induced by a variety of chemical and physical stressors (Morimoto, 2008). It has been reported that HSP70 expression in the intestine is upregulated by intrauterine growth retardation, and the overexpression of HSP70 can protect epithelial cells against physiological and pathological stress and maintain the morphology and function of the gut (Zhong et al., 2010). HSP70 has been shown to protect intestinal epithelial cells from toxic agents and ulcerogenic conditions in the gastrointestinal mucosa, as those cells are susceptible to injury from various stressors and play an important role in digestion, absorption, and metabolism of nutrients (Kojima et al., 2003; Liedel et al., 2011). Similar to HSP70, HSP90 is a molecular chaperone that may exhibit functions associated with preserving the structural integrity of cells following exposure to stress (Li and Buchner, 2013; Yu et al., 2008; Zhang et al., 2018). The results of our experiment are not similar to those of previous studies: We found a decline in the expression of HSP70 and HSP90 in the duodenum and jejunum after both high-dose and low-dose DBP exposures. Previous studies have found minor histological lesions in the hearts of heat-stressed broilers, including enlarged intracellular spaces and slight granular degeneration, and the positive staining for HSPs was distinctly low in the cytoplasm of granular degenerated areas (Yu et al., 2008). The decreasing crypt depth, villus height, and goblet cell number in the duodenum and jejunum of mice exposed to DBP imply that the morphological changes in small intestinal epithelial cells may have an adverse effect on the expression of HSP70 and HSP90; thus, the function of HSP70 and HSP90 may be suppressed by severe damage.

Conclusions

In this investigation, we found that short-term exposure of adult mice to DBP via intragastric administration increases reproductive organ indexes, promotes the growth of villi in the duodenum, and has an adverse effect on the structure of the jejunum. DBP-induced damage to the structure of the epithelium of the small intestine was accompanied by a stress response characterized by a marked increase in HSP27 expression, which was maintained at a high level in mice exposed to both high-dose and low-dose DBPs. These results indicate that elevated HSP27 levels in the duodenum and jejunum may be an important marker for acute DBP exposure and that HSP27 may act as a protective protein involved in intestinal mucosa repair. However, there was a decline in the expression of HSP70 and HSP90 in the duodenum and jejunum in both high-dose and low-dose groups. This suggests that morphological changes in small intestinal epithelial cells may have an adverse effect on the expression of HSP70 and HSP90 and that the function of HSP70 and HSP90 may be suppressed by severe damage.

Footnotes

Authors’ note

Dr Caiyan will deal with correspondence at all stages of refereeing and publication, also post-publication.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a grant from the Scientific Innovation Team Project of Ningbo (no. 2015C110018).

ORCID iD

Jimian Yu

Caiyan Li

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