Blood and organ distribution of mono (2-ethylhexyl) phthalate (MEHP) in rats exposed to di (2-ethylhexyl) phthalate (DEHP) via diet

Di(2-ethylhexyl) phthalate (DEHP) undergoes hydrolysis in the gastrointestinal tract when orally administered to rats, and is mainly distributed in the body as mono(2-ethylhexyl) phthalate (MEHP). MEHP is thought to be involved in testicular and hepatotoxicity. Therefore, clarifying the distribution of MEHP in blood and organs after oral administration of DEHP is important. In this study, male Sprague-Dawley rats were fed 1% and 2% DEHP diets for two weeks and then examined the distribution of MEHP and DEHP concentrations in blood and organs. The following results were obtained.  MEHP concentrations in the 1% group were plasma: 16.7-40.8 μg/ml, testis: 0.9-3.2 μg/g, liver: 21.5-43.6 μg/g, kidney: 6.8-9.6 μg/g and MEHP concentrations in the 2 %group were plasma: 58.5-147.4 μg/ml, testis: 10.5-30.0 μg/g, liver: 44.2-97.1 μg/g,


Chemicals and Animal Diet
DEHP was purchased from Wako pure chemical industries Ltd. (Osaka, Japan).The chemical purities of DEHP and were found to be >97%.CE-2 diets (Clea, Tokyo, Japan) containing DEHP by 1 or 2 w/w% were prepared by Oriental Yeast Company (Chiba, Japan).MEHP was purchased from Tokyo Kasei Kogyo Co., Ltd.(Tokyo, Japan).All other chemicals were the highest grade from commercial sources.

Animals and Ethics
Male Sprague-Dawley rats aged three-week-old purchased from Charles River (Kanagawa, Japan) were housed at the Laboratory Animal Center of Kagawa University.They were acclimated at 22-24 °C and 50-60% relative humidity with a 12-h light/dark cycle.

Experimental Design
Five-week-old rats weighing 166 ± 4 g were divided into control and treatment groups consisting of 6 animals.The treatment group consumed 1% (w / w) DEHP diet or 2% (w / w) DEHP diet for two weeks.At the end of the experiment, rats were sacrificed by ether anesthesia.The testes, liver and kidneys were removed and weighed.Cardiac blood samples were collected in heparinized tubes and plasma was separated from whole blood by centrifugation at 1500 g.Plasma and organs were frozen at -40 °C until MEHP measurement.

Plasma and organ MEHP Measurement
The concentrations of MEHP in plasma and testis were measured by high performance liquid chromatograph (HPLC).
Analytical procyedure and equipment are as follows, 200 µl of 1M NaOH was mixed with 50 µl of plasma, 850 µl of acetonitrile was added, and the mixture was vigorously mixed, followed by ultrasonication for 10 minutes to extract DEHP and MEHP.Thereafter, 10 μl of phosphoric acid was added for neutralization, and the supernatant after centrifugation was subjected to HPLC analysis.For the organs, 300-500 mg of the homogenate was added with 4 times the amount of 1M NaOH and mixed with a homogenizer.
The HPLC analysis conditions were as follows.

Statistics
Results were expressed as means ± standard deviations (SD).Statistical analyses were performed using one-way ANOVA procedure followed by Tukey HSD tests to detect difference between groups.Differences were regarded as significant at P < 0.05.

HPLC analysis
DEHP and MEHP were added to rat plasma at 100 μg/ml and to organs at 100 μg/g, respectively, and the recovery rates were estimated to be 90-100%.The detection limit for MEHP was 0.1μg/g (or 0.1μg/ml).Chromatograms of HPLC analysis are shown in Figure 1.No interfering peaks were detected at the elution positions of MEHP and DEHP in the HPLC analysis of blood and organs.

Animal experiment
The DEHP dose estimated from food intake and average body weight during the dosing period was 0.9 g/kg/day in the 1% group and 1.6 g/kg/day in the 2% group.
Table 1 shows the body weight and organ weight of each group after the DEHP administration experiment.In the 1% group, significant liver hypertrophy was observed, but there was no difference in testis weight and body weight compared to the control.On the other hand, the 2% group showed marked reduction in testis weight, body weight suppression, and liver hypertrophy.
Tables 2 and 3 show the concentrations of MEHP and DEHP in the plasma, testis, liver and kidney of the 1% and 2% groups.The MEHP concentration in the 1% group was plasma: 16.7-40.8μg/ml, testis: 0.9-3.2μg/g, liver: 21.5-43.6μg/g, kidney: 6.8-9.6 μg/g, and plasma in the 2% group.: 58.5-147.4μg/ml,testis: 10.5-30.0μg/g,liver: 44.2-97.1μg/g,kidney: 18.6-56.7μg/g.The average MEHP concentration in the 2% group was higher than that in the 1% group, and by organ, the liver was the highest, followed by the kidney, and the testis was the lowest.On the other hand, DEHP was detected in the range of 1-20 μg/g (μg/ml) in plasma, testis, liver and kidney in both the 1% and 2% groups, but most of them were 2 μg/g (μg/ml) or less, and the kidney showed a slightly higher value than the others.
Figure 2 shows the relationship between the MEHP plasma concentration and each organ concentration.Testis, liver, and kidney MEHP concentrations were all strongly correlated with the plasma MEHP concentration.In addition, the relationship between the MEHP concentration in the testis and the testicular weight, or the plasma MEHP concentration and the testicular weight, both showed a strong negative correlation (Figure 3).

Discussion
Most of DEHP orally administered to rats is hydrolyzed by lipase in the gastrointestinal tract, and the produced monoester MEHP is rapidly absorbed into the body and distributed to organs [7,8].MEHP is mainly involved in toxicity such as testicular atrophy and liver hypertrophy [1][2][3][4][5][6].MEHP is known as a peroxisome proliferator that induces rodent hepatocarcinogenesis [16][17][18].MEHP has also been reported to be a reproductive toxicant that disrupts Sertoli cell function and causes germ cell apoptosis.Therefore, concentrations of MEHP in blood and organs is important in DEHP toxicity.
In this study, MEHP and DEHP concentrations in the blood and organs of male Sprague-Dawley rats were measured after 2 weeks of treatment with 1% and 2% DEHP diets.Mean MEHP concentrations in blood and organs corresponded to DEHP dose, but with considerable inter-individual variability.When looking at the distribution concentration in each organ, the highest was in the liver, followed by the kidney, and the lowest in the testis.Previous studies [8,9] have shown that orally administered DEHP and its metabolites are distributed in large amounts in the liver and kidneys, with little distribution in the testis and brain, which is consistent with the present results.From these results, it was found that considerable concentration differences occur in the distribution of MEHP both by individual and by organ.On the other hand, DEHP was detected in the range of 1-20 μg/g (μg/ml) in plasma, testis, liver and kidney in both the 1% and 2% groups, but most of them were 2 μg/g (μg/ml) or less, and there was no particular relationship between the average DEHP concentrations in blood and organs and the dose of DEHP.Sjoberg et al. [3] detected 48-152 μg/ml of MEHP in the plasma of 25-, 40-, and 60-day-old rats orally administered 1.0 g/kg of DEHP per day for 14 days, but DEHP was not detected.In this experiment, 1% and 2% DEHP were administered for 2 weeks./day.Our results are close to those of Sjoberg et al. [3], probably because the doses were almost the same.Either way.This experiment reconfirmed that orally administered DEHP is mainly distributed in the blood and organs as a monoester MEHP.
Next, it was clarified that MEHP concentration in testis, liver, and kidney showed a strong linear correlation with plasma MEHP concentration.From this, it seemed possible to estimate the amount of MEHP distribution in each organ by measuring the plasma MEHP concentration.
Furthermore, testicular toxicity of DEHP is thought to be related to its metabolite, MEHP [9].A strong negative correlation was observed for both.These results suggest that the degree of testicular toxicity induced by oral administration of DEHP is dependent on the MEHP concentration in the plasma or the MEHP concentration in the target organ, the testis.After reaching the testis, MEHP damages the Sertoli cells in the seminiferous tubules by disrupting the vimentin filaments [12,13], and as a result, the mechanism of germ cell apoptosis is induced [14,15], which is thought to lead to testicular atrophy.Therefore, it is inferred that the relationship observed in this study reflects the dose-effect relationship between MEHP and germ cell apoptosis.Based on these findings, it seemed possible to some extent to evaluate testicular toxicity by measuring plasma MEHP concentrations.

Conclusion
Diets containing 1% and 2% DEHP were administered to male SD rats for 2 weeks, and the concentration distribution of MEHP and DEHP in blood and organs was investigated.The following results were obtained.

Figure 2 Figure 3
Figure 2 Relationship between plasma MEHP concentration and organ concentration

Table 2
MEHP concentrations in plasma and organs of the rats fed 1 or 2% DEHP-containing diet for two weeks

Table 3
DEHP concentrations in plasma and organs of the rats fed 1 or 2% DEHP-containing diet for two weeks