Endocrine adverse effects of mono(2-ethylhexyl) phthalate and monobutyl phthalate in male pubertal rats

Mono(2-ethylhexyl) phthalate (CAS No. 4376-20-9, 97 % purity) and monobutyl phthalate (CAS No. 131-70-4, 97 % purity) as well as the following kits – alanine aminotransferase (ALT) activity assay (catalogue No. MAK052), aspartate aminotransferase (AST) activity assay (catalogue No. MAK055), urea assay (catalogue No. MAK266), triglyceride quantification (catalogue No. MAK006), bromocresol purple albumin assay (catalogue No. MAK125), creatinine assay (catalogue No. MAK080), total protein kit (catalogue No. TP0100), and glucose assay kit (catalog No. CBA086) – were obtained from Sigma-Aldrich (Steinheim, Germany). To measure insulin levels, we used the insulin rat ELISA Kit (Thermo-Fisher Scientific, Waltham, USA). Rat islet cell antibody (catalogue No. BG-RAT11369) was obtained from Novatein Biosciences (Woburn, MA, USA), and rat insulin antibody (catalogue No. RAB0904) from Sigma-Aldrich.

Six-week-old Wistar albino (Rattus norvegicus) male rats (N=66), each weighing 150 –200 g were obtained from the Kobay Laboratory of Experimental Animals (Ankara, Turkey). They were housed in bisphenol A (BPA)-free polycarbonate cages (dimensions 20×40×22 cm) under standard laboratory conditions (22.5±1.5 °C temperature, 47.1±1 % relative humidity, 12:12 h light-dark cycle) and had free access to tap water and standard pellet food throughout the experiment.

The study was approved by the Hacettepe University Experimental Animals Ethics Committee (2020/06-04).

The study was designed as a 28-day subchronic toxicity study. The age of animals and duration of exposure were adjusted to the recommendations of the US EPA Endocrine Disruptor Screening and Testing Advisory Committee (9).

For the experiment, a total of 66 rats were randomly divided into eleven groups of six. Five groups were receiving five different MEHP doses or five different MBP doses, and the control group was receiving 1 mL of corn oil (used as phthalate solvent) by oral gavage every morning for 28 days. Exposed groups were receiving MEHP or MBP in daily doses of 25, 50, 100, 200, and 400 mg/kg bw by gavage. These doses are based on the no-observed-adverse-effect level (NOAEL) and the lowest-observed-adverse-effect level (LOAEL) of the parent phthalates. The NOAEL for DEHP is 5.8 mg/kg bw a day, and the LOAEL 29 mg/kg bw a day (10, 11). The NOAEL of DBP is 19.9 mg/kg bw a day, and the LOAEL 52 mg/kg bw a day (12). Since literature does not report NOAELs and LOAELs for the MEHP and MBP metabolites used in the experiment, the dose of 25 mg/kg bw a day was chosen as the lowest dose in the experimental protocol, taking into account toxicologically relevant values for DEHP and DBP, which should be between the NOAEL and LOAEL. Finally, our experimental protocol consisted of five daily doses, where 25 and 50 mg/kg bw of MEHP and MBP represented low doses, 100 mg/kg bw of MEHP and MBP represented a moderate dose, and 200 and 400 mg/kg bw of MEHP and MBP represented high doses.

The amount of consumed food and water was measured every morning before dosing. Animals were inspected daily to assess survival, clinical signs of toxicity, behavioural impairments, and body weight changes. Body weight of each animal (expressed in grams) was measured at the beginning and end of the experiment. The experiment was terminated after the final gavage at the end of day 28. Animals were humanely euthanized by cervical dislocation under ether anaesthesia.

The blood for biochemical analysis was taken from the hearts with a sterile syringe and put into heparinised vacutainers to prevent clotting. To obtain serum, tubes were centrifuged at 3000 g and +4 °C for 25 min. At the end of centrifugation, serum aliquots were taken to measure ALT and AST, urea, total protein, glucose, albumin, and creatinine levels using commercial measurement kits specified above. The analyses were performed on an automatic SMT-120V chemistry analyser (Chengdu Seamaty Technology, Chengdu, China).

Blood samples for haematological analyses were also taken from the heart as described above. Samples were analysed on the same day using an automatic haematology analyser (DH36, Dymind Biotechnology Co., Shenzhen, China) that simultaneously measured leukocytes (count), neutrophils (%), lymphocytes (%), monocytes (%), red blood cell (count), haemoglobin, haematocrit, mean red blood cell haemoglobin (MCH), mean red blood cell haemoglobin concentration (MCHC), mean corpuscular volume (MCV), thrombocytes (count), and platelets (volume).

Serum samples were also used in immunological assays to determine insulin levels and to detect islet cell and insulin antibodies. These measurements were performed on an ELISA reader (Shimadzu UV-1800 UV-Vis spectrophotometer, Kyoto, Japan) using commercial kits.

During necropsy, liver, kidney and pancreas were dissected and weighed. Some parts of tissue samples were fixed in Bouin solution for 8 h and put into 70 % ethanol. Other parts were fixed in 10 % formalin for 24 h, passed through the ethanol and xylene series, and then embedded in paraffin. Paraffin blocks were cut into 5 μm thick sections using a microtome (Accu-Cut^® SRM^™ 200 Sakura Finetek, Tokyo, Japan). The slides were stained with haematoxylin and eosin and examined under a light microscope (Zeiss Axio Scope A1, Jena, Germany) using the ZEN imaging software (Zeiss, Jena, Germany). Histopathological findings were photographed at magnifications of 200× and 400×.

The obtained results are reported as means ± standard deviations. Statistical analyses were performed using MedCalc^® (MedCalc Software Ltd., Ostend, Belgium). Normal distribution of variables was tested with the Kolmogorov-Smirnov test, while further comparisons were made with one-way analysis of variance (ANOVA) and post-hoc Tukey’s test. Comparisons between groups of variables that did not have normal distribution were made with the Kruskal-Wallis H test and the Mann-Whitney U test. Fisher’s exact test was used to compare the frequencies of histopathological lesions found in the tissues. The level of significance was set to p<0.05.

There were no differences in water consumption between the MEHP-treated groups and control. However, food consumption significantly increased in rats receiving MEHP doses of 200 and 400 mg/kg bw a day compared to control and the groups receiving MEHP doses of 25 and 50 mg/kg bw a day (Table 1).

The MBP-exposed groups showed similar findings (Table 2).

Even though food intake was higher in the treated groups, we found no significant treatment-related increase in body weights in either MEHP or MBP groups (Tables 3 and 4, respectively).

As for individual organs, mean liver weight was significantly higher in groups receiving MEHP doses of 100, 200, and 400 mg/kg bw a day than control. Kidney weights did not differ significantly. Pancreas weight significantly decreased in rats treated with MEHP doses of 200 and 400 mg/kg bw a day compared to control and groups exposed to MEHP doses of 25, 50, 100 mg/kg bw a day (Table 3).

Similar to MEHP, rats receiving MBP doses starting with 100 mg/kg bw a day had significantly higher liver weight than control, whereas kidney weights did not differ. Absolute pancreas weight significantly decreased in the group treated with the MBP dose of 400 mg/kg bw a day compared to control. We also noticed a significant decrease in relative pancreas weight in the group receiving the MBP dose of 200 mg/kg bw a day (Table 4).

Compared to control, ALT, AST, triglyceride, and glucose levels significantly increased in rats treated with MEHP doses of 100, 200, and 400 mg/kg bw a day, whereas total protein, albumin, and urea levels significantly dropped in those receiving 200 and 400 mg/kg bw a day of MEHP (Table 5).

MBP findings were similar to those of MEHP; ALT and AST levels rose significantly with 100, 200, and 400 mg/kg bw a day, both compared to control and the groups treated with lower doses (Table 6). Glucose and triglycerides also increased in the rats receiving MBP doses of 200 and 400 mg/kg bw a day compared to control and the 25 mg/kg bw a day group. Significant drops in total protein, albumin, creatinine, and urea were also observed at the highest MBP doses.

The group receiving the highest MEHP dose had significantly higher MCV than control and the other dose groups, while haemoglobin, leukocyte, and monocyte counts were significantly lower. Other blood parameters did not differ significantly between the MEHP-exposed groups and control (Table 7).

Treatment with MBP showed similar findings (Table 8), as the highest dose significantly increased MCV and decreased haemoglobin compared to the other groups. Other blood parameters did not differ significantly.

The highest two doses of both MEHP and MBP significantly lowered insulin levels compared to the other groups (Tables 9 and 10, respectively). However, all blood islet and insulin antibody tests were negative in all groups (Table 11).

Figures 1 –3 show respective typical histological changes in the liver, kidney, and pancreas of rats receiving either MEHP or MBP. Liver changes included mononuclear cell infiltration and minimal congestion (Figure 1B, E, and F), sinusoidal degeneration (Figure 1C), cytoplasmic dissolution (Figure 1D), and a necrotic region (1G). Kidney changes included separation in parietal and visceral leaves in glomerulus and minimal congestion (Figure 2B and C) and congestion with atrophic glomerulus and tubular degeneration (Figure 2D and E). Changes in the pancreas included congestion and extensive destruction (Figure 3B) and degeneration and vacuolar cytoplasm in the islets of Langerhans (Figure 3C, D, and E).

Figure 1

Figure 2

Lesions in all organs are visible in more than a half of treated animals per group receiving the MEHP dose of 100 mg/kg a day or higher (Table 12). Similar are the findings for MBP, save for the pancreas, where more than a half of the animals were affected at the two highest doses (Table 13).