Di-n-pentyl Phthalate Research Articles

Phthalate migration potential in vacuum-packed fish.

Phthalates or phthalate esters (PAEs) have become a serious concern due to their toxicity and risks of migration from contact materials to food matrices and the environment. The aim of this study is to monitor the possible migration potential of PAEs in pelagic fish stored in vacuum packaging depending on the storage time and to determine the polyethylene polymers. In order to achieve this goal, sea bass (Dicentrarchus labrax) and anchovy fish (Engraulis encrasicolus) were randomly packaged in vacuum bags and then stored for 90days. Phthalate content was determined by GC/MS technique in the muscle tissue of each fish species at certain periods (0, 30, and 90days) of storage, and on the first day in the packaging material and fish meat. As a result of the analysis performed in µ-Raman spectroscopy, no microplastics were detected in both fish species' meats. FTIR spectroscopy results of the packaging material determined nylon in the chemical content of the packaging material before processing. It has been determined that the chemical composition of the packaging used in the vacuum packaging process is affected by the temperature, depending on the storage period, and different polymer types are formed in the processed package material. It was determined that the dominant PAE homologues were Di-n-pentyl phthalate (DPENP) in both fish meat and Di-(2-ethylhexyl)-phthalate (DEHP) in the package. However, during storage, Dibutylphthalate (DBP) became dominant in anchovies and DPENP became dominant in sea bass, differing according to fish species and storage time.

Exposure to dipentyl phthalate in utero disrupts the adrenal cortex function of adult male rats by inhibiting SIRT1/PGC-1α and inducing AMPK phosphorylation.

Di-n-pentyl phthalate (DPeP) is an endocrine-disrupting phthalate plasticizer. The objective of this study was to investigate the effect of DPeP on adrenocortical function in adult male rats following in utero exposure. DPeP (0, 10, 50, 100, and 500 mg/kg/day) was administered by gavage to pregnant Sprague-Dawley rats from gestational day 14 to 21. The morphology and function of the adrenal cortex in 56-day-old male offspring were studied. DPeP at 100 and 500 mg/kg/day significantly reduced serum aldosterone levels and at 500 mg/kg/day markedly reduced corticosterone and adrenocorticotropic hormone levels. DPeP at 10-500 mg/kg markedly reduced the thickness of zona glomerulosa without affecting the thickness of zona fasciculata. DPeP significantly downregulated the expression of Agtr1a, Mc2r, Scarb1, Cyp11a1, Hsd3b1, Cyp21, Cyp11b1, Cyp11b2, Nr5a1, Nr4a2, and Bcl2 genes as well as their proteins. DPeP at 500 mg/kg/day significantly increased phosphorylated AMPK, while DPeP at 100 mg/kg/day and higher doses reduced phosphorylated AKT1 and total SIRT1 level. DPeP at 100 and 500 μM markedly induced reactive oxygen species and apoptosis in H295R cells after 24 h of culture. In conclusion, in utero exposure to DPeP disrupts adrenocortical function of the adult male offspring by (1) increasing AMPK phosphorylation and decreasing AKT1 phosphorylation and SIRT1 levels, (2) reducing adrenocorticotropic hormone levels, and (3) possibly inducing oxidative stress and apoptosis.

Spatiotemporal occurrence of phthalate esters in stormwater drains of Hong Kong, China: Mass loading and source identification

Urban stormwater is an important pathway for transporting anthropogenic pollutants to water bodies. Phthalate esters (PAEs) are endocrine disruptors owing to their estrogenic activity and potential carcinogenicity and their ubiquitous presence has garnered global interest. However, their transportation by urban stormwater has been largely overlooked. This study, for the first time, investigated 15 PAEs in stormwater from six major stormwater drains in the highly urbanized Hong Kong, a major metropolitan city in China. The results showed that PAEs were ubiquitous in the stormwater of Hong Kong, with total concentrations (∑15PAEs) spanning from 195 to 80,500 ng/L. Bis(2-n-butoxyethyl) phthalate (DBEP), diisopentyl phthalate (DiPP), dicyclohexyl phthalate (DCHP) and di-n-pentyl phthalate (DnPP) were detected in stormwater for the first time. Spatial variations in PAEs were observed among different stormwater drains, possibly due to the different land use patterns and intensities of human activities in their respective catchments. The highest and lowest levels of ∑15PAEs were found in Kwai Chung (3860 ± 1960 ng/L) and the Ng Tung River (672 ± 557 ng/L), respectively. Additionally, significantly higher concentrations of ∑15PAEs in stormwater were found in the wet season (2520 ± 2050 ng/L) than in the dry season (947 ± 904 ng/L). Principal component analysis classified domestic and industrial origins as two important sources of PAEs in the stormwater of Hong Kong. Stormwater played a crucial role in transporting PAEs, with an estimated annual flux of 0.705–29.4 kg. Thus, possible stormwater management measures were proposed to protect the receiving environment and local ecosystems from stormwater.

Phthalate metabolites in urine of children and adolescents in Germany. Human biomonitoring results of the German Environmental Survey GerES V, 2014-2017.

During the population representative German Environmental Survey of Children and Adolescents (GerES V, 2014-2017) 2256 first-morning void urine samples from 3 to 17 years old children and adolescents were analysed for 21 metabolites of 11 different phthalates (di-methyl phthalate (DMP), di-ethyl phthalate (DEP), butylbenzyl phthalate (BBzP), di-iso-butyl phthalate (DiBP), di-n-butyl phthalate (DnBP), di-cyclohexyl phthalate (DCHP), di-n-pentyl phthalate (DnPeP), di-(2-ethylhexyl) phthalate (DEHP), di-iso-nonyl phthalate (DiNP), di-iso-decyl phthalate (DiDP) and di-n-octyl phthalate (DnOP)). Metabolites of DMP, DEP, BBzP, DiBP, DnBP, DEHP, DiNP and DiDP were found in 97%-100% of the participants, DCHP and DnPeP in 6%, and DnOP in none of the urine samples. Geometric means (GM) were highest for metabolites of DiBP (MiBP: 26.1μg/L), DEP (MEP: 25.8μg/L), DnBP (MnBP: 20.9μg/L), and DEHP (cx-MEPP: 11.9μg/L). For all phthalates but DEP, GMs were consistently higher in the 3-5 years old children than in the 14-17 years old adolescents. For DEHP, the age differences were most pronounced. All detectable phthalate biomarker concentrations were positively associated with the levels of the respective phthalate in house dust. In GerES V we found considerably lower phthalate biomarker levels than in the preceding GerES IV (2003-2006). GMs of biomarker levels in GerES V were only 18% (BBzP), 23% (MnBP), 23% (DEHP), 29% (MiBP) and 57% (DiNP) of those measured a decade earlier in GerES IV. However, some children and adolescents still exceeded health-based guidance values in the current GerES V. 0.38% of the participants had levels of DnBP, 0.08% levels of DEHP and 0.007% levels of DiNP which were higher than the respective health-based guidance values. Accordingly, for these persons an impact on health cannot be excluded with sufficient certainty. The ongoing and substantial exposure of vulnerable children and adolescents to many phthalates confirms the need of a continued monitoring of established phthalates, whether regulated or not, as well as of potential substitutes. With this biomonitoring approach we provide a picture of current individual and cumulative exposure developments and body burdens to phthalates, thus providing support for timely and effective chemicals policies and legislation.

Iron-based magnetic molecular imprinted polymers and their application in removal and determination of di-n-pentyl phthalate in aqueous media.

Iron-based magnetic molecular imprinted polymers (Fe@SiO2@MIP) were synthesized for highly selective removal and recognition of di-n-pentyl phthalate (DnPP) from water. Well-defined core-shell Fe@SiO2 nanoparticles (less than 70 nm) were decorated on MIPs reticular layers to endow DnPP-MIPs with magnetic property for the first time. Five other phthalic acid esters including dimethyl phthalate, diethyl phthalate, dipropyl phthalate, di-n-butyl phthalate and di-iso-octyl phthalate were used to investigate the adsorptive selectivity to DnPP. The designed experiments were carried out to explore the adsorption kinetics, isotherms and thermodynamics and the results demonstrated that the adsorption was a spontaneous, exothermal and physical adsorption process. The materials were proved to be excellent adsorbents in removal of DnPP with an adsorption capacity as high as 194.15 mg g−1 in optimal condition. Furthermore, a magnetic solid phase extraction with Fe@SiO2@MIP coupled to high-performance liquid chromatography method was successfully developed for the determination of DnPP, and the proposed method achieved a good linear range of 0.5–250 µg l−1 with a correlation coefficient (R2) of 0.999 and low limit of detection (LOD) of 0.31 µg l−1. These materials exhibited excellent capacity in removal and highly sensitive identification of DnPP from aqueous environment samples, and opened a valuable direction for developing new adsorbents for the removal and enrichment of important pollutants.

Phthalate 3종에 대한 경피투과 특성 연구

Objectives: Phthalates are used in a large variety of products including as coatings of pharmaceutical tablets, film formers, stabilizers, dispersants, emulsifying agents, and suspending agents. They have been the subject of great public concern in recent years. The extensive uses of this material have attracted attention and issues regarding its safety have been raised. Methods: In this study, three types of phthalate skin permeation were studied using matrixes such as ointments, creams and lotions in vitro. The absorption of phthalate diesters [Dimethyl phthalate (DMP), Din-propyl phthalate (DPP) and Di-n-pentyl phthalate (DNPP)] using film former has been measured in vitro through rat skin. Epidermal membranes were set up in Franz diffusion cells and their permeability to PBS measured in order to establish the integrity of the skin before the phthalates were applied to the epidermal surface. Results: Absorption rates for each phthalate ester were determined and permeability assessment made to quantify any irreversible alterations in barrier function due to contact with the esters. Types of phthalate in vitro experimental results quickly appeared in the following order DMP > DPP ≥ DNPP. Conclusions: In the experimental results, lotion> cream> ointment, and the permeation rate of lotion with a great amount of moisture was the fastest. Skin permeation rate is generally influenced by the chemical characteristics of a given chemical, such as molecular weight and lipophilicity. As the esters became more lipophilic and less hydrophilic, the rate of absorption decreased.

Separation of phthalates by cyclodextrin modified micellar electrokinetic chromatography: Quantitation in perfumes

A new CE method has been developed for the simultaneous separation of a group of parent phthalates. Due to the neutral character of these compounds, the addition of several bile salts as surfactants (sodium cholate (SC), sodium deoxycholate (SDC), sodium taurodeoxycholate (STDC), sodium taurocholate (STC)) to the separation buffer was explored showing the high potential of SDC as pseudostationary phase. However, the resolution of all the phthalates was not achieved when employing only this bile salt as additive, being necessary the addition of neutral cyclodextrins (CD) and organic modifiers to the separation media. The optimized cyclodextrin modified micellar electrokinetic chromatography (CD-MEKC) method consisted of the employ of a background electrolyte (BGE) containing 25mM β-CD-100mM SDC in a 100mM borate buffer (pH 8.5) with a 10% (v/v) of acetonitrile, employing a voltage of 30kV and a temperature of 25°C. This separation medium enabled the total resolution of eight compounds and the partial resolution of two of the analytes, di-n-octyl phthalate (DNOP) and diethyl hexyl phthalate (DEHP) (Rs~0.8), in only 12min. The analytical characteristics of the developed method were studied showing their suitability for the determination of these compounds in commercial perfumes. In all the analyzed perfumes the most common phthalate was diethyl phthalate (DEP) that appeared in ten of the fifteen analyzed products. Also dimethyl phthalate (DMP), diallyl phthalate (DAP), dicyclohexyl phthalate (DCP), and di-n-pentyl phthalate (DNPP) were found in some of the analyzed samples.

Urinary and serum metabolites of di- n-pentyl phthalate in rats

Di- n-pentyl phthalate (DPP) is used mainly as a plasticizer in nitrocellulose. At high doses, DPP acts as a potent testicular toxicant in rats. We administered a single oral dose of 500 mg kg −1 bw of DPP to adult female Sprague–Dawley rats ( N = 9) and collected 24-h urine samples 1 d before and 24- and 48-h after DPP was administered to tentatively identify DPP metabolites that could be used as exposure biomarkers. At necropsy, 48 h after dosing, we also collected serum. The metabolites were extracted from urine or serum, resolved with high performance liquid chromatography, and detected by mass spectrometry. Two DPP metabolites, phthalic acid (PA) and mono(3-carboxypropyl) phthalate (MCPP), were identified by using authentic standards, whereas mono- n-pentyl phthalate (MPP), mono(4-oxopentyl) phthalate (MOPP), mono(4-hydroxypentyl) phthalate (MHPP), mono(4-carboxybutyl) phthalate (MCBP), mono(2-carboxyethyl) phthalate (MCEP), and mono- n-pentenyl phthalate (MPeP) were identified based on their full scan mass spectrometric fragmentation pattern. The ω − 1 oxidation product, MHPP, was the predominant urinary metabolite of DPP. The median urinary concentrations (μg mL −1) of the metabolites in the first 24 h urine collection after DPP administration were 993 (MHPP), 168 (MCBP), 0.2 (MCEP), 222 (MPP), 47 (MOPP), 26 (PA), 16 (MPeP), and 9 (MCPP); the concentrations of metabolites in the second 24 h urine collection after DPP administration were significantly lower than in the first collection. We identified some urinary metabolic products in the serum, but at much lower levels than in urine. Because of the similarities in metabolism of phthalates between rats and humans, based on our results and the fact that MHPP can only be formed from the metabolism of DPP, MHPP would be the most adequate DPP exposure biomarker for human exposure assessment. Nonetheless, based on the urinary levels of MHPP, our preliminary data suggest that human exposure to DPP in the United States is rather limited.

Enzymatic hydrolysis of structurally diverse phthalic acid esters by porcine and bovine pancreatic cholesterol esterases

A weak hydrolyzing activity against bis (2-ethylhexyl) phthalate (DEHP) was discovered in a commercial crude lipase (EC 3.1.1.3) preparation from porcine pancreas. DEHP was hydrolyzed to mono (2-ethylhexyl) phthalate (MEHP) not by a pancreatic lipase but by a cholesterol esterase (CEase, EC 3.1.1.13), a trace contaminant in the crude lipase preparation. Enzymatic hydrolysis of phthalic acid esters (PAEs), suspected to be endocrine-disrupting chemicals, was investigated using CEases from two species of mammals and a microorganism. Eight structurally diverse PAEs, namely diethyl phthalate (DEP), di- n-propyl phthalate (DPrP), di- n-butyl phthalate (DBP), di- n-pentyl phthalate (DPeP), di- n-hexyl phthalate (DHP), DEHP, n-butyl benzyl phthalate (BBP), and dicyclohexyl phthalate (DCHP), were hydrolyzed to their corresponding monoesters by both porcine and bovine pancreatic CEases, while a microbial CEase from Pseudomonas sp. had no hydrolyzing activity against these PAEs. The hydrolysis experiments with bovine pancreatic CEase (50 U) indicated complete hydrolysis of every PAE (5 μmole) except for BBP and DCHP within 15 min; BBP and DCHP were hydrolyzed within 30 min and 6 h, respectively. The rates of PAE hydrolysis could be affected by the bulkiness of alkyl side chains in the PAEs. This study provides important evidence that mammalian pancreatic CEases, such as those from porcine and bovine sources, are potential enzymes for nonspecific degradation of structurally diverse PAEs.

Acute Adverse Effects of the Indenopyridine CDB‐4022 on the Ultrastructure of Sertoli Cells, Spermatocytes, and Spermatids in Rat Testes: Comparison to the Known Sertoli Cell Toxicant Di‐n‐pentylphthalate (DPP)

Acute effects of CDB-4022 on testicular ultrastructure were determined. Rats were treated orally with vehicle or a maximally effective single dose of CDB-4022 or Di-n-pentylphthalate (DPP). Preserved testes were processed for transmission electron microscopy. Sertoli and germ cells of vehicle-treated rats demonstrated normal morphological characteristics. Disruption of Sertoli cell ultrastructure was apparent in CDB-4022-treated rats by 3 hours. A decrease in the presence of nucleoli, an increase in the amount and diameter of swollen smooth endoplasmic reticulum, and decreases in cytoplasmic ground substance were observed. The severity of these degenerative effects increased at 6 and 12 hours: Vacuoles were apparent; increased cellular debris, swollen mitochondria, and phagocytic structures were observed; and membranes became more disorganized. Similar ultrastructural changes were observed in the Sertoli cells of DPP-treated rats. By 3 hours, spermatocytes and spermatids were adversely affected by CDB-4022 treatment with swelling of the nuclear envelope. The Step 8 spermatids were especially noteworthy; chromatin was more diffuse and rarefied, the nuclear envelopes were incomplete or broken, and the position of the spermatid nucleus within the cell and relative to Sertoli cell cytoplasm was unusual. Fusion of spermatids to form giant cells was observed by 12 hours. CDB-4022 acts acutely on Sertoli cells to induce marked cellular rarefaction and degeneration, but not necrosis. A rapid and direct effect of CDB-4022 on spermatocytes and spermatids was observed. The antispermatogenic activity of CDB-4022 appears to be a consequence of direct effects on Sertoli and germ cells.

Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells.

Phthalates are widely used in industry and cause public concern since they have genomic estrogenic-like effects via estrogen receptors. We previously found that some phthalates have nongenomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChRs) in bovine chromaffin cells. In this study, we investigated the effects of eight phthalates on the calcium signaling of human nAChR by using human neuroblastoma SH-SY5Y cells. All eight phthalates, with different potency, have inhibitory roles on the calcium signaling coupled with human nAChR, but not muscarinic acetylcholine receptors (mAChRs). For inhibition of human nAChR, the strongest to weakest potencies were observed as di-n-pentyl phthalate (DPP) --> butyl benzyl phthalate (BBP) --> di-n-butyl phthalate (DBP) --> dicyclohexyl phthalate (DCHP) --> di-n-hexyl phthalate (DHP) --> di-(2-ethyl hexyl) phthalate (DEHP) --> di-n-propyl phthalate (DPrP) --> diethyl phthalate (DEP). The potencies of phthalates were associated with their structures such that the most effective ones had dialkyl group carbon numbers of C4 or C5, with shorter or longer numbers resulting in decreased potency. At as low as 0.1 microM, DPP, DBP, BBP, DCHP and DHP significantly inhibited the calcium signaling of human nAChR. The IC50 of phthalates on human nAChR, ranging from 0.32 to 7.96 microM, were 10-50 lower than those for bovine nAChR. We suggest that some phthalates effectively inhibit the calcium signaling of human nAChR, and these nongenomic effects are cause for concern.

Octanol/Water Partition Coefficients and Water Solubilities of Phthalate Esters

Measurements of the octanol/water partition coefficients (Kow) and water solubilities of di-n-octyl phthalate (DnOP) and di-n-decyl phthalate (DnDP) by the slow-stirring method are reported. The water solubility was also measured for di-n-hexyl phthalate (DnHP). The log Kow values of dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DnBP), di-n-pentyl phthalate (DnPP), and DnHP, determined in a previous study, and the log Kow values of DnOP determined again in this study were correlated against the number of carbons in the phthalate side chain. The straight line regression equation (log Kow = 0.979 + 0.615, r2 = 0.993) was used to extend the estimate for a carbon number 10; that is, log Kow = 10. 41 for DnDP. The slow-stirring log Kow = 8.83 ± 0.05 reported here for DnDP suggests that the decrease in solubility of the phthalate in the water layer is nonlinear when the side chain carbon length increases beyond eight carbons. Similarly, the straight line regression equation derived from correlation of the measured water solubilities for DnOP and DnHP reported here and the literature values for DMP, DEP, and DnBP against side chain carbon number predicted a 0.005 μg/L water solubilty for DnDP. The measured water solubility of DnDP (0.22 ± 0.05 μg/L) reported here was 44-fold greater.

The Influence of Phthalate Esters on Leydig Cell Structure and Function in Vitro and in Vivo

Phthalate esters are widely used in the manufacture of plastics and have been shown to cause testicular toxicity, purportedly, by targeting the Sertoli cell alone. Recent evidence, however, indicates that a paracrine control exists between Sertoli and Leydig cells and the breakdown of one component of this relationship is therefore detrimental to normal function. However, no data that explore the influence of testicular toxins on Leydig cell structure and function have been published hitherto. The preliminary studies reported here were initiated to test the hypothesis that phthalate intoxication may adversely alter Leydig cell structural and functional integrity. Four phthalate esters, namely, di(2-ethylhexyl) phthalate (DEHP), di- n-pentyl phthalate (DPP), di- n-octyl phthalate (DOP), and diethyl phthalate (DEP) were investigated in vivo and their monoesters (MEHP, MPP, MOP, and MEP, respectively) in vitro for indications of Leydig cell toxicity in the rat. Rats were dosed by oral gavage with 2 g phthalate diester/kg/day in corn oil vehicle for 2 days, while Leydig cell primary cultures were incubated with 1000 μ M monoester for 2 hr. Light and electron microscopy were undertaken to determine the type and degree of any changes. Phthalate esters exerted a direct effect on Leydig cell structure and function (as determined by testosterone output) with correlation of the in vitro and in vivo effects of MEHP (DEHP) and MOP (DOP). No effects on Leydig cell structure or function were seen with MPP (DPP), although Sertoli cell cytoplasmic rarefaction and vacuolation were observed in vivo. DEP produced Leydig cell ultrastructural alterations in vivo. We conclude that individual phthalate esters may exert effects on both Sertoli and Leydig cells or one cell type alone.

Di- n-pentyl phthalate-induced inflammatory changes in the rat testis are accompanied by local production of a novel lymphocyte activating factor

Phthalate esters (PAE) are plasticizers in polyvinyl chloride plastics used, for example, in package material for medical solutions. PAE exposure is associated with testicular damage that primarily affects Sertoli cells, and is concomitant with leukocyte infiltration into the testicular interstitium. We have demonstrated that the rat testis constitutively produces a lymphocyte activating factor (LAF) resembling interleukin-1α, and originating from Sertoli cells. The testicular interleukin-1-like factor (tIL-1) has a relative molecular mass ( M r) of 17,000 (17 k) and an isoelectric point (p I) of 5.7. In the present study we have measured testicular LAF activity after exposure to di- n-pentyl phthalate (DPP) in 40-day-old rats. We found a large increase in LAF bioactivity which was evident already 3 h after a single oral dose of DPP. The increase was maximal 9–12 h after exposure, and had decreased toward the control level at 24 h. The increased activity was found to be at least partly due to the induction of a novel LAF with M r 38,000 and p I 4.5. Morphological examination confirmed earlier results with an interstitial leukocyte infiltration 6 h after DPP exposure. The identity of the novel LAF and its functional relation to testicular inflammation remain to be established.

Comparison of changes in serum androgen binding protein with germinal epithelial damage and infertility induced by di- n-pentyl phthalate

Androgen binding protein (ABP), produced by Sertoli cells and released into seminiferous tubules and blood, was measured in the serum of di- n-pentyl phthalate (DPP)-treated rats as a potential index of germinal epithelial damage. A single oral dose of DPP (0, 0.25, 1.0, or 2.0 g/kg body wt in corn oil) was given to four groups of 110 Fischer 344 rats; 10 rats per group were killed weekly for 10 weeks. Effects of treatment on serum ABP were then compared with effects on other reproductive endpoints. Treatment did not produce any significant effect on body weight or weights of liver, kidney, prostate, and seminal vesicles. In high-dose rats, serum ABP values more than doubled 2 days after injection, remained significantly elevated for 3 weeks, then fell and remained significantly below control values from Week 4 through Week 10. Accordingly, 95% of the rats in this group showed >50% of the seminiferous tubules degenerated, decreased epididymal sperm density, reduced testicular and epididymal weights, and up to 97% morphologically abnormal sperm. In medium-dose rats, serum ABP increased up to 48% during the first week, returned to control values by Week 2, and remained at control levels thereafter. Of these rats, 20% showed 20–50% degenerated tubules, decreased sperm density, reduced testicular and epididymal weights (which were not always statistically significant), and up to 23% abnormal sperm morphology. In low-dose rats, serum ABP levels were similar to those of controls, and the other parameters, except sperm density, also remained unchanged. To examine the effects of DPP on fertility, a second group of rats was exposed in an identical manner [gavaged once with DPP in corn oil (0, 0.25, 1.0, and 2.0 g/kg body wt)], then mated to untreated females at 3, 6, and 10 weeks postexposure. DPP at 2 (but not 1.0 or 0.25) g/kg caused a significant reduction in pregnancies and live pups and a significant increase in preimplantation loss. Histopathology of the testis in the first experiment suggested a very slow recovery. Therefore, controls and high-dose rats in the mating trial were killed 14, 18, and 30 weeks after dosing and the germinal epithelium was evaluated histologically. All high-dose animals showed testicular lesions typical of phthalate ester exposure and the epithelium did not recover within 30 weeks. These results indicate that serum ABP levels changed rapidly after a single dose of DPP and also that DPP caused adverse effects on male reproductive function. Serum ABP may be useful as a peripheral marker of severe Sertoli cell toxicity.

Effects of mono-(2-ethylhexyl) phthalate and mono- n-pentyl phthalate on the ultrastructural morphology of rat Sertoli cells in Sertoli/germ cell co-cultures: Correlation with the in vivo effects of di- n-pentyl phthalate

The effects of mono-(2-ethylhexyl) phthalate (MEHP) and mono- n-pentyl phthalate (MPP) on the ultrastructure of Sertoli cells in primary co-cultures of rat Sertoli and germ cells has been examined and compared with the in vivo effects of di- n-pentyl phthalate (DPP) on Sertoli cells in the rat. MEHP and MPP produced changes in the configuration of the plasma membrane, resulting in long thin cytoplasmic processes or filopodia. This was accompanied by changes in microfilament distribution and an increased density of ribosomes, smooth endoplasmic reticulum and the Golgi complex. Changes were qualitatively similar for both monoesters, but MEHP was effective at lower concentrations than MPP. In vivo, DPP produced extensive convolutions in the basal plasma membranes separating adjacent Sertoli cells and retraction of the lateral processes surrounding germ cells. The similarity of the membrane changes in vivo and in vitro suggests a comparable response by the Sertoli cell in the two systems. Furthermore, the similarity of the in vitro response to the two monoesters suggests a shared mechanism of toxicity.

The ultrastructural effects of di- n-pentyl phthalate on the testis of the mature rat

A sequential morphological study has been carried out to examine the ultrastructural effects of di- n-pentyl phthalate (DPP) on the mature rat testis. A single oral dose of 2.2 g DPP/kg body wt was administered, and testes, perfuse-fixed 3–48 hr after dosing, were examined by transmission electron microscopy. By 3 hr, rarefaction of the basal Sertoli cell cytoplasm was seen and the basal plasma membranes separating adjacent Sertoli cells were thrown into a series of convoluted profiles with the appearance of interdigitating cell processes. The subjacent ectoplasmic specializations that normally face these membranes were disrupted, and by 12 hr the inter-Sertoli junctions showed numerous membrane discontinuities. The lateral processes of Sertoli cell cytoplasm, which separate germ cells, showed retraction and fragmentation, resulting in direct contact between adjacent germ cells or the isolation of germ cells unapposed by Sertoli cell plasma membrane. In addition, the ectoplasmic specializations associated with Sertoli-spermatid and Sertoli-pachytene spermatocyte junctions were often disrupted or absent. The mitochondria in the Sertoli cells were enlarged and, in some tubules, increased in number. The changes seen were restricted to tubules in the successive stages XI–XIV, I, and II of the spermatogenic cycle. Elongating spermatids (steps 12–15) showed cytoplasmic condensation and vacuolation by 12 hr and were necrotic by 24 hr. A small proportion of zygotene and early pachytene spermatocytes showed necrosis by 24 hr after dosing. By 48 hr, the cytoplasmic rarefaction and convoluted plasma membranes had regressed and ectroplasmic specializations had reformed along Sertoli-Sertoli junctions.

Aspects of the testicular toxicity of phthalate esters.

Di(2-ethylhexyl) phthalate (DEHP) produced seminiferous tubular atrophy and reductions in seminal vesicle and prostate weight in 4-week-old, but not in 15-week-old rats. Di-n-pentyl phthalate (DPP) did produce atrophy in the older rats but this developed more slowly than in young animals. Coadministration of testosterone or gonadotrophins did not protect against phthalate-induced testicular toxicity but did partly reverse the depression of seminal vesicle and prostate weight. Secretion of seminiferous tubule fluid and androgen binding protein by the Sertoli cells was markedly suppressed within 1 hr of a dose of DPP or mono-2-ethylhexyl phthalate (MEHP) in immature rats. This occurred less rapidly in mature rats. [14C]Mono-n-pentyl phthalate and [14C]MEHP penetrated the blood testis barrier only to a very limited extent. These findings and the early morphological changes in the Sertoli cells produced by DPP suggest that phthalate esters may act initially to cause Sertoli cell injury, the subsequent loss of germ cells occurring as a consequence of this. Some features of the testicular lesion could be reproduced in primary cocultures of rat Sertoli and germ cells. Structure activity studies with a range of phthalate monoesters showed good agreement between the induction of germ cell detachment in culture and testicular toxicity in vivo. Three metabolites of MEHP (metabolites V, VI, and IX) were much less toxic in culture than MEHP itself, suggesting that the latter may be the active testicular toxin from DEHP.

Species differences in the testicular toxicity of phthalate esters

Oral administration of di- n-butylphthalate (DBP) produced uniformly severe seminiferous tubular atrophy in rats and guinea pigs but caused only focal atrophy in mice. Hamsters showed no testicular changes with DBP and only minor changes in response to di-(2-ethylhexyl)phthalate (DEHP) and di- n-pentylphthalate (DPP). The rate of intestinal monohydrolysis of DEHP was significantly slower in hamsters than in rats and this may be important, as mono-(2-ethylhexyl)phthalate (MEHP) did cause focal seminiferous tubular atrophy in hamsters. However, mono- n-butylphthalate (MBP) had no such effect. The decrease in testicular zinc concentration and enhancement of urinary zinc excretion produced in rats by DEHP and DPP was not observed in hamsters. Thus, species differ widely in their sensitivity to the testicular toxicity of phthalate esters.