Induction of xenobiotic-metabolizing enzymes and peroxisome proliferation in rat liver caused by dietary exposure to di(2-ethylhexyl)phosphate

Abstract

1. Exposure of rats to 1% or 3% (w/w) di(2-ethylhexyl)phosphate in the diet for five days results in two- to three-fold inductions of liver cytosolic epoxide hydrolase activity and microsomal cytochrome P-450 content. Cytochromes P-450b + e were induced 20- to 35-fold, but no increase was observed in cytochrome P-450c.

2. Considerably smaller effects were obtained on NADPH-cytochrome c reductase, microsomal epoxide hydrolase and microsomal cytochrome b₅ content, and there was no effect on cytosolic glutathione transferase activity, under the same conditions.

3. A dramatic increase in cyanide-insensitive palmitoyl-CoA oxidation and total mitochondrial protein, together with smaller increases in total catalase and cytochrome oxidase activities, were observed after treatment with di(2-ethylhexyl)phosphate, indicating that this compound causes proliferation of both peroxisomes and mitochondria.

4. It is suggested that the induction of cytosolic epoxide hydrolase and the proliferation of peroxisomes may be related processes.     

Induction of peroxisome proliferation in rat liver by dietary treatment with 2,2,4,4,6,8,8-heptamethylnonane

1. Exposure of rats to 1% (w/w) of 2,2,4,4,6,8,8-heptamethylnonane in the diet for 2 weeks resulted in marked induction of liver peroxisome proliferation as judged from electron micrography, elevated activities of hepatic catalase (36%), cyanide-insensitive palmitoyl-CoA oxidase (10-fold), carnitine acetyl transferase (9.6-fold), lauric acid hydroxylase (12.4-fold), and the induction of the 80 K protein in SDS-polyacrylamide gel electrophoresis (4.1-fold). 2. 2,2,4,4,6,8,8-Heptamethylnonane dicarboxylic acid, a non-beta-oxidizable fatty acid, was detected as the major metabolite in the liver, an example of an unmetabolizable lipophilic anion as a peroxisome proliferator.     

Comparison of tris(2-ethylhexyl) phosphate and di(2-ethylhexyl) phosphoric acid toxicities in a rat 28-day oral exposure study

Tris(2-ethylhexyl) phosphate (TEHP, CAS no. 78-42-2) is a plasticizer and a flame retardant, while di(2-ethylhexyl) phosphoric acid (DEHPA, CAS no. 298-07-7) is an oil additive and extraction solvent. Publicly-available information on repeated exposure to these two related organophosphate compounds is fragmentary. Hence, adult male and female Fischer rats were exposed to TEHP (300, 1000 and 3000 mg/kg body weight [BW]/day) or DEHPA (20, 60 and 180 mg/kg BW/day) by gavage for 28 consecutive days, to assess and compare their toxicities. Although significantly impaired BW gains and evidence of TEHP enzymatic hydrolysis to DEHPA were observed only in males, exposures to the highest TEHP and DEHPA doses often resulted in similar alterations of hematology, serum clinical chemistry and liver enzymatic activities in both males and females. The squamous epithelial hyperplasia and hyperkeratosis observed in the non-glandular forestomach of rats exposed to the middle and high DEHPA doses were most likely caused by the slightly corrosive nature of this chemical. Although tubular degeneration and spermatid retention were observed only in the testes of males exposed to the highest TEHP dose, numerous periodic acid-Schiff stained crystalline inclusions were observed in testis interstitial cells at all TEHP dose levels. No-observed-adverse-effect levels for TEHP and DEHPA are proposed, but the lower serum pituitary hormone levels resulting from TEHP and DEHPA exposures and the perturbations of testicular histology observed in TEHP-treated males deserve further investigation. Improved characterization of the toxicity of flame retardants will contribute to better informed substitution choices for legacy flame retardants phased-out over health concerns.     

Hepatic peroxisome proliferation and hypolipidemic effects of di(2-ethylhexyl)phthalate in neonatal and adult rats

To determine the relative sensitivity of suckling rats as compared to adults to the effects of di(2-ethylhexyl) phthalate (DEHP), five daily oral doses of 0, 10, 100, 1000, or 2000 mg DEHP/kg body weight were given to male Sprague-Dawley rats beginning at 6, 14, 16, 21, 42, and 86 days of age. Twenty-four hours after the last dose, rats were sacrificed and plasma cholesterol and triglyceride levels and the activities of the hepatic peroxisomal enzymes, palmitoyl CoA oxidase and carnitine acetyltransferase, were determined. Suckling rats (1-3 weeks of age) suffered severe growth retardation at doses of 1000 mg/kg and death at 2000 mg/kg while older rats only showed decreased weight gain at 2000 mg/kg. Of particular interest was the lethality at doses of 1000 mg/kg at 14 days of age but not at 16 days or at other ages. Increases in relative liver weight and hepatic peroxisomal enzyme activities were similar in all age groups except the 14-day old group in which the increases were greater. Relative kidney weight was increased in 21-, 42-, and 86-day-old rats at the highest doses but not in younger rats. Hypolipidemia was observed only in 21-, 42-, and 86-day-old rats at doses of 1000 and 2000 mg/kg, while elevated plasma cholesterol levels were observed in 6- and 14-day-old rats at the 1000 mg/kg dose, possibly due to the dietary differences between suckling and weaned rats. The results suggest that neonatal and suckling rats are more sensitive to the lethal and growth retardation effects of DEHP than are adult rats, but the hepatic peroxisome proliferation is similar at all ages with the exception of a greater increase at 14 days of age.     

The influence of di(2-ethylhexyl)phthalate on protein turnover in rat liver

Treatment of rats with the plasticizer di(2-ethylhexyl)phthalate increases liver weight and leads to proliferation of mitochondria and peroxisomes. Using in vivo labelling with [3H]leucine, an increased rate of incorporation into the total protein of mitochondria and microsomes was observed. The half-lives of proteins in subcellular fractions were determined using [35S]methionine labelling. The half-lives for the total protein of mitochondria, microsomes, and supernatant were increased from 6 to 25 days, from 3.5 to 5.5 days and from 2.5 to 5 days upon treatment with phthalate esters. Experiments with [14C]guanidino-L-arginine indicated that some reutilization of [35S]methionine occurred, but this did not influence the results substantially. It appears that phthalate esters increase protein synthesis and decrease protein breakdown, the former effect being of greater importance.     

Investigation of the potential for binding of di(2-ethylhexyl) phthalate (DEHP) and di(2-ethylhexyl) adipate (DEHA) to liver DNA in vivo

It was the aim of this investigation to determine whether covalent binding of di(2-ethylhexyl) phthalate (DEHP) to rat liver DNA and of di(2-ethylhexyl) adipate (DEHA) to mouse liver DNA could be a mechanism of action contributing to the observed induction of liver tumors after lifetime feeding of the respective rodent species with high doses of DEHP and DEHA. For this purpose, DEHP and DEHA radiolabeled in different parts of the molecule were administered orally to female rats and mice, respectively, with or without pretreatment for 4 weeks with 1% unlabeled compound in the diet. Liver DNA was isolated after 16 hr and analyzed for radioactivity. The data were converted to a covalent binding index, CBI = (micromoles of substance bound per mole of DNA nucleotides)/(millimoles of substance applied per kilogram body weight), in order to allow a quantitative comparison also with other carcinogens and noncarcinogens. Administration of [¹⁴C]carboxylate-labeled DEHP to rats resulted in no measurable DNA radioactivity. The limit of detection, CBI < 0.02 was about 100 times below the CBI of compounds where an observable tumor-inducing potential could be due to genotoxicity. With [¹⁴C]- and [³H]DEHP labeled in the alcohol moiety, radioactivity was clearly measurable in rat liver DNA. HPLC analysis of enzyme-degraded or acid-hydrolyzed DNA revealed that the natural nucleosides or purine bases were radiolabeled whereas no radioactivity was detectable in those fractions where the carcinogen-modified nucleoside or base adducts are expected. The respective limits of detection were at 0.07 and 0.04 CBI units for the ¹⁴C and ³H labels, respectively. The experiments with [¹⁴C]- and [³H]DEHA, labeled in the alcohol moiety and administered to mice, revealed a minute radioactivity of <50 dpm/mg liver DNA, too little to allow a nucleoside analysis to determine that fraction of the radioactivity which had been incorporated via biosynthesis. Expressed in the CBI units, values of 0.05 to 0.15 for ¹⁴C and 0.01 to 0.12 for ³H resulted. Determination of the level of ¹⁴CO₂ expiration revealed a linear correlation with the specific activity of DNA. Experiments with 2-ethyl[1-¹⁴C]hexanol performed with both rats and mice allowed the conclusion that most if not all DEHA radioactivity in mouse liver DNA was due to biosynthetic incorporation. A maximum possible true DNA binding by DEHA must be below CBI 0.01. Pretreatment of the animals with unlabeled compound had no effect on the DNA radioactivities in either species. The present negative data, in conjunction with other negative short-term tests for mutagenicity, strongly indicate that covalent interaction with DNA is highly unlikely to be the mode of tumorigenic action of DEHP and DEHA in rodents.     

Gene expression changes induced in mouse liver by di(2-ethylhexyl) phthalate

Increasing chemical use necessitates a better understanding of how pollutants, such as di(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator and a phthalate plasticizer, affect human health. To understand the effects of DEHP exposure, we utilized microarray technology to identify novel DEHP targets in livers of male C57BL/6 mice treated with 1.0% dietary DEHP for 13 weeks. We identified 51 DEHP-regulated genes; these genes are involved in, but not limited to, peroxisome proliferation, xenobiotic detoxification, oxidative stress response, immune function, steroid hormone metabolism, testis development, and pheromone transport. The reproductive toxicity mechanism of DEHP may be due to its effects on steroid hormone metabolism and sexual development. Confirmation of microarray results with Northern blots demonstrated that both low- and high-dose DEHP treatments altered the expression of genes associated with testis development and steroid hormone synthesis. Vanin-1, a regulator of testis development, was upregulated after exposure to 0.2 and 1.0% DEHP by 1.8- and 2.9-fold, respectively. Several genes involved in hormone metabolism were also regulated by DEHP. 11betaHSDI was downregulated 1.8- and 3.1-fold by 0.2 and 1.0% DEHP, respectively, while HSD3b5 was suppressed similarly by 0.2% DEHP, 1.5-fold, and more severely by 1.0% DEHP, 8.0-fold. Interestingly, food restriction had a similar effect to DEHP on several genes, including HSD3b5. In addition, steroidogenic gene cyp7B1 was downregulated while phospholipid transfer protein and cyp2B9 were induced. Genes peripherally associated with steroid hormones were also affected: ALDH3, GSTtheta2, and Id2. Collectively, our data validate the concern for DEHP as a reproductive developmental toxicant.     

Peroxisome proliferation due to di (2-ethylhexyl) adipate, 2-ethylhexanol and 2-ethylhexanoic acid

The dose-response relationships for peroxisome proliferation due to Di (2-ethylhexyl) adipate (DEHA), 2-ethylhexanol (EH), 2-ethylhexanoic acid (EHA) have been investigated in rats and mice. Linear dose-response relationships were observed for induction of cyanide-insensitive palmitoyl CoA oxidation (PCO), used as a enzyme marker of peroxisome proliferation, by DEHA, EH and EHA in both species. Relative liver weights were also increased in a dose related manner. On a molar basis, DEHA was twice as potent as EH or EHA which were equipotent and PCO was stimulated to a greater extent in male mice than in rats or female mice. At doses above 8 mmol/kg/day, EH was toxic to rats (both sexes) and similarly EHA at 13.5 mmol/kg/day lead to the death of female rats. In a attempt to explain the species difference in carcinogenicity of DEHA previously reported, we also used Fischer 344 rats and B6C3F1 mice. DEHA administration (2.5 g/kg/day) to Fischer 344 rats and B6C3F1 mice lead to toxicity in female rats. Relative liver weights were increased in a dose related fashion by DEHA administration to both rats and mice, PCO but not catalase was markedly increased (up to 15 fold in male rats). Light microscopy examination indicated some glycogen loss, a dose related hypertrophy and increased eosinophilia in both rats and mice. Electron microscopy confirmed peroxisome proliferation accompanied by a marked reduction of lipid in the centrilobular hepatocytes. These data suggest EHA to be the proximate peroxisome proliferator derived from DEHA. These data indicate a higher sensitivity for Fischer 344 rats than B6C3F1 mice to hepatic peroxisome proliferation due to DEHA and ratio of PCO activity and catalase activity data suggest that more hydrogen peroxide (H₂O₂) could escape from peroxisomes in male Fischer 344 rats than B6C3F1 mice. These data obtained with B6C3F1 mice and Fischer 344 rats are not agreement with the carcinogenicity bioassays previously reported showing an incidence of hepatic tumours only in B6C3F1 mice.     

Identification of the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate

A primary rat hepatocyte culture system was utilized to determine the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate (DEHP). DEHP was administered to rats and the urinary metabolites were identified and isolated. The major metabolites were those resulting from initial omega- or omega - 1-carbon oxidation of the mono(2-ethylhexyl) phthalate (MEHP) moiety. These metabolites, together with MEHP and 2-ethylhexanol, were added to primary rat hepatocyte cultures and the effect on peroxisomal enzyme activity was determined. The omega-carbon oxidation products [mono(3-carboxy-2-ethylpropyl) phthalate (I) and mono(5-carboxy-2-ethylpentyl) phthalate (V)] and 2-ethylhexanol produced little or no effect on CN- -insensitive palmitoyl-CoA oxidation (a peroxisomal marker). MEHP and the omega - 1-carbon oxidation products [mono-(2-ethyl-5-oxohexyl) phthalate (VI) and mono(2-ethyl-5-hydroxyhexyl) phthalate (IX)] produced a large (7- to 11-fold) induction of peroxisomal enzyme activity. Similar structure-activity relationships were observed for the induction of cytochrome P-450-mediated lauric acid hydroxylase and increase in cellular coenzyme A content. This identification of the proximate proliferators will aid in the elucidation of the mechanism by which DEHP causes proliferation of peroxisomes in the rodent liver. Oral administration of MEHP (150 or 250 mg/kg) to male guinea pigs did not produce hepatic peroxisome proliferation. Addition of MEHP (0 to 0.5 mM) or one of the "active" proliferators in the rat (metabolite IX, 0 to 0.5 mM) to primary guinea pig hepatocyte cultures also failed to produce an induction of peroxisomal beta-oxidation. Possible reasons for this species difference are discussed.     

Induction of cytochrome P450 and other drug metabolizing enzymes in rat liver following dietary exposure to Aroclor 1254

Selected drug metabolizing activities were measured in female F344/NCr rats exposed to graded dietary concentrations of Aroclor 1254 (1 to 1000 ppm) for 7 days or to lower concentrations of Aroclor (1 to 10 ppm) for up to 28 days. Following the 7-day exposure, the hepatic O-dealkylation of ethoxyresorufin (ETR), mediated primarily by cytochrome P450IA, was increased 60-, 10-, and 4-fold by 33, 10, and 3 ppm Aroclor, respectively. In rats exposed to 10 and 3 ppm Aroclor for 28 days, this activity was increased approximately 30- and 10-fold, respectively. Hepatic ETR O-dealkylase activities correlated with Aroclor concentrations in the livers of exposed rats (r = 0.99, p less than 0.01). Although the O-dealkylation of benzyloxyresorufin was highly increased by 7-days dietary exposure to 1000 ppm Aroclor, the levels of Aroclor necessary for detection of induction were substantially higher than those required for detection of ETR O-dealkylase induction. Examination of the non-P450-mediated drug metabolizing activities, epoxide hydrolase and DT-diaphorase, similarly showed limited (approximately 10-fold) increases. In contrast, aldehyde dehydrogenase (benzaldehyde, NADP+) activity was highly increased (greater than 40-fold) at 1000 ppm, however this activity was increased to only a limited extent at lower Aroclor concentrations (e.g. approximately 3-fold at 33 ppm). These results support the potential use of cytochrome P450 activities as potential biomarkers for environmental exposure to PCBs and related compounds.     

低剂量邻苯二甲酸二(2-乙基)己酯对老年大鼠前列腺的促增生作用

目的评价环境暴露剂量的邻苯二甲酸二(2-乙基)己酯(DEHP)对老年大鼠前列腺的促增生作用及机制。方法 32只1.5岁龄老年雄性SD大鼠随机分成4组,每组8只,分别ig给予DEHP(30,90和270μg·kg~(-1))或溶媒,每天1次,连续4周。于末次给药24 h后将大鼠麻醉,而后(1)腹主动脉采血,采用ELISA检测血清中睾酮(T)、雌二醇(E2)和泌乳素(PRL)水平;(2)处死后取前列腺,分叶,称重、测量体积,计算脏器系数;(3)制作前列腺组织病理切片,HE染色后用显微镜观察组织形态改变,并利用显微图像分析软件分析前列腺上皮高度变化。结果与溶媒对照组比较,DEHP 270μg·kg~(-1)组前列腺系数、背侧前列腺质量和背侧前列腺系数均显著增加(P<0.05);DEPH各剂量组腹侧前列腺上皮高度明显升高(P<0.01);DEHP 270μg·kg~(-1)组背侧前列腺上皮高度明显升高(P<0.01);DEHP各剂量组E2,PRL和T水平均无显著改变,但DEHP 30和270μg·kg~(-1)组E2/T比值显著增加(P<0.05)。结论低剂量DEHP对老年大鼠前列腺具有促增生作用,该作用可能与其影响内源性激素的相对水平有关。     

Induction of propranolol metabolism in isolated rats hepatocytes treated by di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP).

Blood lines of polyvinyl chloride (PVC) for hemodialysis usually contain di(2-ethylhexyl) phthalate (DEHP) as a plasticizer. Previous studies show that 1 mg/kg of this plasticizer can leach into the blood during one dialysis session. It is rapidly metabolized in the liver. Mono(2-ehtylhexyl) phthalate (MEHP), its main metabolite can be detected as well. After oral administration to rodents, both compounds caused a variety of adverse biological effects such as testicular atrophy, peroxisome proliferation and hepatic peroxisomal enzyme induction. Male wistar rats were treated intraperitoneally by DEHP and MEHP using twice the dose of that involved in human exposure during a dialysis session. Propranolol metabolism by hepatocytes was investigated after fresh isolation from treated and untreated rats by means of reverse phase HPLC. The choice of propranolol as a substrate was made because of its rather quick liver metabolisation. Phenobarbital was chosen in the study as a reference of enzymatic inducer to evaluate the inducing effect of DEHP and MEHP. Propranolol was metabolized by the hepatocytes of both treated and untreated rats. Hepatocytes isolated from rats treated by phenobarbital, MEHP and DEHP were shown to have a higher speed constant of metabolism indicating a rapid metabolism of propranolol. Under these conditions, in fact, propranolol metabolisation was found to be respectively 6, 2.7, 2 times faster than the propranolol metabolisation of untreated rats. The hypothesis that DEHP and MEHP are enzymatic inducers, particularly cytochrome P450 (CYP) inducers of the xenobiotics metabolism on the intact liver after IP administration has become been found to be valid. The results obtained in this study confirm the value of isolated hepatocytes as an in vivo drug metabolism predictive model.     

Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate

Di(2-ethylhexyl) phthalate (DEHP) is a well-known testicular toxicant inducing adverse effects in androgen responsive tissues. Therefore, di(2-ethylhexyl) adipate (DEHA) is currently being evaluated as a potential substitute for DEHP. Similarities in structure and metabolism of DEHP and DEHA have led to the hypothesis that DEHA can modulate the effects of DEHP. Wistar rats were gavaged with either vehicle, DEHP (300 or 750 mg/kg bw/day) or DEHP (750 mg/kg bw/day) in combination with DEHA (400 mg/kg bw/day) from gestation day (GD) 7 to postnatal day (PND) 17.

Decreased anogenital distance (AGD) and retention of nipples in male offspring were found in all three exposed groups. Dosed males exhibited decreased weights of ventral prostate and m. levator ani/bulbocavernosus. Histopathological investigations revealed alterations in testis morphology in both juvenile and adult animals. The litter size was decreased and postnatal mortality was increased in the combination group only, which is likely a combined effect of DEHP and DEHA. However, no combination effect was seen with respect to antiandrogenic effects, as males receiving DEHP in combination with DEHA did not exhibit more pronounced effects in the reproductive system than males receiving DEHP alone.     

Chronic peroxisome proliferation and hepatomegaly associated with the hepatocellular tumorigenesis of di(2-ethylhexyl)phthalate and the effects of recovery.

This study compared the levels of cell proliferation and peroxisome proliferation in rodent liver with tumor incidence, to provide more information on the relationship between these events following chronic exposure. Fischer 344 rats were treated with 0, 100, 500, 2500, or 12,500 ppm DEHP, and B6C3F1 mice were treated with 0, 100, 500, 1500, or 6000 ppm DEHP in the diet for up to 104 weeks. Additional groups of rats and mice received the highest concentration for 78 weeks and then the control diet for an additional 26 weeks (recovery groups). Animals were terminated at weeks 79 and 105 for histopathologic examination. Elevated palmitoyl CoA oxidation activity and higher liver-to-body weight ratios were observed for the 2500- and 12,500-ppm groups of rats, and for the 500-, 1500-, and 6000-ppm groups of mice at Week 105. No increase in palmitoyl CoA oxidation activity was evident in the recovery group, and relative liver weights were near control levels following recovery. No hepatic cell proliferation was detected at Weeks 79 or 105 in either species although preliminary data indicated that cell proliferation did occur within the first 13 weeks of exposure. A significantly higher incidence of hepatocellular tumors was only observed for the 2500- and 12,500-ppm group and its recovery group of rats, and for the 500-, 1500-, and 6000-ppm groups and the recovery group of mice. The tumor incidences were reduced for the recovery groups compared with the groups fed DEHP continuously for 104 weeks. The data indicate that high levels of peroxisome proliferation and hepatomegaly are associated with DEHP hepatocarcinogenesis in rodent liver, and that the tumorigenic process may be arrested by cessation of DEHP treatment, suggesting that extended treatment with DEHP acts to promote tumor growth.     

Pesticide exposure and genetic variation in xenobiotic-metabolizing enzymes interact to induce biochemical liver damage

Metabolic activation of pesticides in the liver may result in highly reactive intermediates capable of impairing various cellular functions. Nevertheless, the knowledge about the effect of pesticide exposure on liver function is still limited. This study assessed whether exposure to pesticides elicits early biochemical changes in biomarkers of liver function and looked for potential gene-environmental interactions between pesticide exposure and polymorphisms of pesticide-metabolizing genes. A longitudinal study was conducted in farm-workers from Andalusia (South Spain), during two periods of the same crop season with different degree of pesticide exposure. Blood samples were taken for the measurement of serum and erythrocyte cholinesterase activities as well as for determining clinical chemistry parameters as biomarkers of liver function. Serum lipid levels were also measured as they may help to monitor the progress of toxic liver damage. A reduction in serum cholinesterase was associated with decreased levels of all clinical chemistry parameters studied except HDL-cholesterol. Conversely, a decreased erythrocyte cholinesterase (indicating long-term pesticide exposure) was associated with increased levels of aspartate aminotransferase and alkaline phosphatase and increased levels of triglycerides, total cholesterol and LDL-cholesterol, but reduced levels of HDL-cholesterol. Changes in liver biomarkers were particularly associated with the PON1_55M/192R haplotype. The obtained results therefore support the hypothesis that pesticide exposure results in subtle biochemical liver toxicity and highlight the role of genetic polymorphisms in pesticide-metabolizing enzymes as biomarkers of susceptibility for developing adverse health effects.     

Effect of testosterone on the testicular atrophy caused by di(2-ethylhexyl)phthalate (DEHP)

The involvement of testosterone in di(2-ethylhexyl)phthalate (DEHP) induced testicular injury has been examined by coadministration of testosterone (1 mg/kg) along with DEHP (2000 mg/kg) daily for 15 days. The coadministration of testosterone and DEHP appears to have prevented the testicular injury as judged by the biochemical and histopathological changes. The sperm count and the activity of the testicular enzymes, gamma-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), sorbitol dehydrogenase (SDH), beta-glucuronidase and acid phosphatase, related with the maturation of sperm, which were significantly altered by DEHP treatment were found to be within normal levels after the combination treatment of DEHP and testosterone. The histopathological studies also showed more or less normal spermatogenic events. The results of this study have suggested the involvement of testosterone in DEHP induced testicular atrophy.     

The individual and combined metabolite profiles (metabolomics) of dibutylphthalate and di(2-ethylhexyl)phthalate following a 28-day dietary exposure in rats

Metabolite profiles (metabolomics) of plasma samples of Wistar rats dosed with di(2-ethylhexyl)phthalate (DEHP – 3000 ppm) and dibutylphthalate (DBP – 150, 1000 and 7000 ppm) were individually determined in 28 days dietary studies. In addition, profiles of combined exposure to 3000 ppm DEHP and either 150, 1000 or 7000 ppm DBP were determined.