Effects of airway exposure to di-(2-ethylhexyl) phthalate on allergic rhinitis

Abstract

Recent epidemiological studies have suggested a positive link between atopy morbidity and exposure to phthalate esters, which are environmental chemicals mainly involved in house dust. Nevertheless, experimental studies applying several allergic in vivo models (in addition to epidemiological studies) are needed to prove the precise correlation between phthalates and facilitation of the allergic response/pathophysiology. Among the phthalate esters, di-(2-ethylhexyl) phthalate (DEHP) has been widely used in flexible polyvinyl chloride products, including vinyl flooring and wall covering, and has been widely suggested to have immunomodulating potential. In the present study, we examined the effects of airway exposure to DEHP on allergen (ovalbumin: OVA)-induced rhinitis in mice. The repeated administration of OVA via an intranasal route induced nasal inflammation characterized by the infiltration of granulocytes (neutrophils and eosinophils) into the nasal cavity. In this experimental setting, DEHP did not exaggerate OVA-related inflammatory pathology. However, local (nasal) IL-13 levels were significantly higher in mice treated with allergen plus DEHP than with allergen alone. Taken together, phthalate esters including DEHP have the potential to exacerbate the allergic milieu in the nasal system, as well as dermal and respiratory systems.     

Adjuvant effect of di-(2-ethylhexyl) phthalate on asthma-like pathological changes in ovalbumin-immunised rats

Pollution of phthalates, including di-(2-ethylhexyl)phthalate (DEHP), is widespread because of their use in plastics and other daily consumer products. Epidemiological studies have shown that the largest source of general population exposure to DEHP is on dietary. Studies have suggested an association between exposure to phthalate plasticisers, and increased prevalence of asthma. To investigate the adjuvant effect of DEHP on asthmatic pathological changes in ovalbumin-immunised rat model. Wistar rats were randomly divided into five groups (eight rats of each group): (1) saline; (2) ovalbumin (OVA); (3) OVA+DEHP 0.7mg·kg^?1·d^?1; (4) OVA+DEHP 70 mg·kg^?1·d^?1; and (5) DEHP 70 mg·kg^?1·d^?1. DEHP treatment groups which were administered to 0.7 mg and 70 mg DEHP/kg/d by gastric gavage for 30d before and during the process of OVA immunisation or saline treatment. The in vivo pulmonary function analysis, bronchoalveolar lavage (BAL) fluid collection and section histological observation were conducted. DEHP exposure could significantly increase airway hyperresponsiveness, airway remolding, and eosinophil infiltration in the OVA-immunised rats. But the DEHP exposure alone group does not show significant airway structural change and inflammatory cell infiltration, compared with control group. DEHP administered by gastric gavage play an adjuvant effect on respiratory systems in ovalbumin-immunised rat model.     

Reactivity of catecholamines and related substances in the mouse lymphoma L5178Y cell assay for mutagens

Using the L5178Y mouse lymphoma cell thymidine kinase locus and the Salmonella his locus assays, the mutagenic potentials of several catecholamines and related compounds were examined. No supplementary metabolic activation systems were used. In the mouse lymphoma assay, the dihydroxybenzenes catechol and hydroquinone had similar and appreciable mutagenic potentials, whereas resorcinol was less active. Derivatives of catechol, such as dopamine and epinephrine, were mutagenic, whereas the related monohydroxylated compounds tyramine and synephrine were inactive. The primary amine, arterenol, and the corresponding secondary amine, epinephrine, induced similar mutagenic responses. Carboxylation of the side chain of dopamine, giving L-dopa, reduced the maximum mutagenic response. The introduction of charged groups directly on to the aromatic ring also reduced mutagenic activity, while an intervening methylene reversed this effect. Thus, 3,4-dihydroxyhydrocinnamic acid was more active than 3,4-dihydroxybenzoic acid. The compound active at the lowest doses was 4-tert-butyl catechol. The activities of these compounds are highly dependent upon substituent groups. Experiments with superoxide dismutase gave circumstantial evidence for some of the mutagenic activity being due to superoxide anion. Active oxygen species might be responsible for some of the observed effects, but this cannot be concluded from the superoxide dismutase experiments. Mutagenic responses in Salmonella were very low but were significant for L-dopa in three strains and for epinephrine and arterenol in one strain. Limited DNA association studies of 14C-dopamine suggest interactions in L5178Y and Salmonella cells and in mouse liver. The mutagenicity of dopamine in L5178Y is reduced by high serum concentrations during the exposure period, while the apparent association with DNA is unaffected.     

High plasma concentrations of di-(2-ethylhexyl)-phthalate in women with endometriosis

BACKGROUND: Emerging evidence suggests a potential role for ubiquitous environmental contaminants in the physiopathology of endometriosis. Di-(2-ethylhexyl)-phthalate (DEHP), the most commonly used plasticizer in flexible polyvinylchloride (PVC) formulations, is a widespread environmental contaminant with potentially adverse effects on fertility in animal models. In the present study, we tested the hypothesis that DEHP and/or and its main metabolite, mono-ethylhexyl phthalate (MEHP), play a role in the pathogenesis of endometriosis. METHODS: Specimens of blood and peritoneal fluid were collected in a group of women with endometriosis (n = 55), and in age-matched control women (n = 24). Concentrations of DEHP and MEHP were measured in plasma and peritoneal fluid by using high performance liquid chromatography (HPLC). Differences between groups were tested using the Fisher's exact test, Wilcoxon-test, and Kruskal-Wallis analysis of variance. RESULTS: Endometriotic women showed significantly higher plasma DEHP concentrations than controls (median 0.57 micro g/ml, interquartile range: 0.06-1.23; values range: 0-3.24 versus median 0.18 micro g/ml, interquartile range: 0-0.44; values range: 0-1.03; P = 0.0047) and 92.6% of them had detectable DEHP and /or MEHP in the peritoneal fluid. No significant differences in either the DEHP/MEHP plasma concentrations (P >/= 0.31) or DEHP/MEHP peritoneal fluid concentrations (P >/= 0.66) were observed in the endometriotic patients as a function of the disease stage at the time of diagnosis. CONCLUSIONS: The present findings showed for the first time an association between DEHP plasma concentrations and endometriosis, suggesting a possible role for phthalate esters in the pathogenesis.     

Multicolor spectral karyotyping of the L5178Y Tk+/- -3.7.2C mouse lymphoma cell line

The L5178Y/Tk+/- -3.7.2C mouse lymphoma cell line is characterized, at the cytogenetic level, by a karyotype involving both numerical and complex structural aberrations. While the karyotype is remarkably normal for a transformed cell line that has been in culture for almost half a century, there are a number of chromosomal alterations that because of their complexity cannot be fully characterized by routine or even high-resolution G-banding studies. Multicolor spectral karyotyping (SKY) was performed on the cell line in anticipation of identifying the previously unresolved chromosome aberrations and confirming interpretations previously identified by banding studies. New chromosome aberrations detected by SKY include numerical aberrations of chromosome 15, duplications of regions of chromosomes 4, 5, 12, and 18, and deletion of chromosome 14. Complex unbalanced translocations involved segments of chromosomes 6, 14, and 15. In total, the SKY technique was able to provide new refined designations on segments of eight different chromosome pairs (4, 5, 6, 9, 12, 14, 15, 18) and identified all three previously unidentified marker chromosomes. This analysis provides an updated standard reference for the karyotype of the L5178Y/Tk+/- -3.7.2C cell line used in the in vitro mouse lymphoma mutation assay.     

Cytogenetic evaluation of di-(2-ethylhexyl)phthalate and its major metabolites in Fischer 344 rats

Di-(2-ethylhexyl)phthalate (DEHP) and its two major metabolites, mono-(2-ethylhexyl)phthalate (MEHP) and 2-ethylhexanol (EH), were evaluated for their ability to induce chromosomal damage in male Fischer 344 rats after oral administration. Dose levels, the highest of which represents one-tenth of the five-day LD50, were based on a preliminary five-day dose-finding study for each test material. The dose levels selected were 5.0, 1.7, and 0.5 ml/kg/day for DEHP; 0.14, 0.05 and 0.01 ml/kg/day for MEHP; and 0.21, 0.07 and 0.02 ml/kg/day for EH. All test materials and vehicle control (corn oil) were administered by gavage for five consecutive days. A triethylenemelamine (TEM)-positive control group received a single intraperitoneal injection of 0.5 mg/kg TEM one day prior to sacrifice. Of the 50 metaphase bone marrow cells examined from each animal, no significant increase in chromatid and chromosome breaks or structural rearrangements were noted for DEHP, MEHP, and EH. In addition, the mitotic index, determined from 100 cells per animal, was unaffected by DEHP, MEHP, or EH. The results of this investigation indicate that DEHP, MEHP, and EH, at these dose levels, did not induce detectable chromosomal aberrations after oral administration.     

The spectral karyotype of L5178Y TK+/− mouse lymphoma cells clone 3.7.2C and factors affecting mutant frequency at the thymidine kinase (tk) locus in the microtitre mouse lymphoma assay

There has been much discussion on acceptable spontaneous mutant frequencies in the mouse lymphoma assay (MLA). This culminated in the International Workshop on Genotoxicity Testing (IWGT) recommended control limits for the microtitre version of 50–170 mutants/10⁶ viable cells, which has now been included in the draft Organization for Economic Co‐Operation and Development guideline for assays investigating mammalian cell gene mutation at the tk locus. Some of the factors affecting mutant frequency have been investigated. It was shown that when culturing methotrexate cleansed TK^+/? cells, a spontaneous mutant frequency of ～100 mutants/10⁶ viable cells was achieved after only 26 doublings. However, after further culturing for ～6 months the spontaneous mutant frequency only gradually increased. Culturing for this time did not affect the karyotype of the cell in so much as the modal chromosome number remained stable. The spontaneous mutant frequency could effectively be manipulated by cleansing with various concentrations of methotrexate. The necessity for using appropriately heat‐inactivated horse serum was confirmed. Finally, following treatment with 4‐nitroquinoline‐N‐oxide, cells did not preferentially survive when plated at high cell densities (1.6 cells plus 2,000 feeder cells/well) versus cells at low density (1.6 cells/well). It was considered that these findings confirm that the dynamics of spontaneous mutant formation in the MLA are complex. However, the karyotype of L5178Y cells is remarkably stable and assuming investigators are using cells with appropriate provenance and good culturing technique, it is clear that the IWGT recommendations are achievable. Environ. Mol. Mutagen. 55:35–42, 2014. © 2013 Wiley Periodicals, Inc.     

Evaluation of the mutagenicity of an N-nitroso contaminant of the sunscreen Padimate O: N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO).

The nitrosamine contaminant, N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO), of the major sunscreen ingredient Padimate O (4-N,N'-dimethylamino-benzoic acid, 2-ethylhexyl ester) was synthesized and tested for mutagenicity in the Salmonella typhimurium and mouse lymphoma L5178Y TK +/- assays. In contrast to the previously reported positive responses in S. typhimurium tester strains TA100 and TA1535 [Loeppky et al., 1991], there were no increases in the number of revertants with strains TA98, TA100, TA1535, and TA1538 in either the Salmonella plate incorporation [Ames et al., 1975] or preincubation [Yahagi et al., 1977] assays. Additional testing with Salmonella, following the modified preincubation procedure [Rogan, 1990] that gave the initial positive response, was also negative. Data from the mouse lymphoma assays were also uniformly negative. During synthesis of NPABAO, small amounts of 4-N,N'-dimethylamino-3-nitrobenzoic acid, 2-ethylhexyl ester (DMANBAO) can be formed. To determine whether the reported positive mutagenicity response of NPABAO could be the result of trace amounts of DMANBAO in the NPABAO, that compound was also synthesized and tested for mutagenicity with Salmonella. Positive responses were obtained with tester strains TA98 and TA 1538 but not with TA100 and TA1535, indicating that DMANBAO was not responsible for the increase in revertants originally reported.     

Evaluation of laser dye mutagenicity using the ames/salmonella microsome test

Twenty-five laser dyes and four analogs were tested for mutagenicity in the Ames/Salmonella test. Seven dyes and two analogs gave positive mutagenic responses with bacterial strains TA1538 and TA98. Of two widely used families of laser dyes (coumarins and rhodamines), four coumarin samples, but none of the rhodamine samples, were mutagenic. All mutagenic compounds require enzyme activation for positive response except two terphenyl analogs, which are mutagenic with or without activation. Using high-performance liquid chromatography (HPLC), it was determined that five mutagenic dye samples had multiple components. The dyes themselves may not be the mutagenic agents in all cases (as with Nile Blue) but may contain impurities that are mutagenic. One dye, adicyanome-thylene (DCM) (≥95% pure), was mutagenic at doses below 0.5 μg/plate on strains TA1538 and TA98. DCM also induced reversions in strains TA96, TA97, TA100, TA102, and TA104, although less efficiently. This study indicates the need for further toxicological testing of these types of compounds. The mutagenic components of these dye mixtures, whether it is the dye or a contaminant, presents a possible hazard to those handling them. Therefore, practices and procedures for the safe handling of specific dyes should be reviewed in light of these findings.     

邻苯二甲酸二乙基己酯对妊娠大鼠激素的影响及锌的拮抗作用

目的通过动物实验研究DEHP对妊娠期大鼠体内性激素的影响及运用锌对DEHP的保护作用。方法将50只雌鼠随机分为5组,即空白对照组、玉米油对照组、DEHP组、锌组、锌+DEHP组。每组与妊娠前后给予相应的药物干预,至妊娠后第19天处死大鼠并取血。送血标本检测FSH、LH、雌二醇、孕酮、睾酮五种性激素的值。结果 DEHP组LH、雌二醇、孕酮、睾酮四种激素水平较各对照组均下降,而FSH反馈性升高,比较差异具有显著性意义(P<0.05)。DEHP+锌组五种性激素水平与对照组相比未见明显改变,没有统计学意义(P>0.05);与DEHP组相比五种性激素水平具有显著性差异,具统计学意义(P<0.05)。结论 DEHP可干扰妊娠期大鼠的内分泌环境,加锌可保护DEHP对机体的损伤。     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay: III. 72 coded chemicals

Seventy-two chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay, using procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Significant responses were obtained with allyl isothiocyanate, p-benzoquinone dioxime, benzyl acetate, 2-biphenylamine HCl, bis(2-chloro-1-methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2-chloroethanol, chlorothalonil, cytarabine.HCl, p,p'-DDE, diazinon, 2,6-dichloro-p-phenylenediamine, N,N-diethylthiourea, diglycidylresorcinol ether, 2,4-dimethoxy aniline.HCl, disperse yellow 3, endosulfan, 1,2-epoxyhexadecane, ethyl acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4'-methylenedianiline.2 HCl, methyl viologen, nickel sulfate.6H2O, 4,4'-oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6-tetrachloro-4-nitro-anisole, 1,1,1,2-tetrachloroethane, trichlorfon, 2,4,6-trichlorophenol, 2,4,5-trimethoxybenzaldehyde, 1,1,3-trimethyl-2-thiourea, 1-vinyl-3-cyclopetene dioxide, vinyl toluene, and ziram. Apart from 2-biphenylamine.HCl, 2-chloroethanol, disperse yellow 3, ethylene thiourea, FD and C yellow number 6, phenol, and 1,1,2-tetrachloroethane, rat liver S9 mix was not a requirement for these compounds. Chemicals not identified as mutagens were acid red, 11-aminoudecanoic acid, boric acid, 5-chloro-o-toluidine, coumaphos, cyclohexanone, decabromodiphenyl oxide, di(2-ethylhexyl)adipate, ferric chloride, fluometuron, melamine, monuron, phenesterin, phthalimide, reserpine, sodium dodecyl sulfate, 4,4-sulfonyldianiline, tetrachloroethylene, and zearalenone. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these were benzyl alcohol, 1,4-dichlorobenzene, phenol, succinic acid-2,2-dimethyl hydrazide, and toluene.     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay. II: 18 coded chemicals

Eighteen chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay by the use of procedures based upon those described by Clive and Spector [Mutat Res 44:269-278, 1975] and Clive et al [Mutat Res 59:61-108, 1979]. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Significant responses were obtained with benzofuran, benzyl chloride, bromodichloromethane, butylated hydroxytoluene, chlorendic acid, o-chlorobenzalmalonitrile, 1,2,3,4-diepoxybutane, dimethyl formamide, dimethyl hydrogen phosphite, furfural, glutaraldehyde, hydroquinone, 8-hydroxyquinoline, and resorcinol. Apart from bromodichloromethane, butylated hydroxytoluene and dimethyl hydrogen phosphite, rat liver S9 mix was not a requirement for the activity of any of these compounds. Chemicals not identified as mutagens were water, tert-butyl alcohol, pyridine, and witch hazel.     

Structure-activity studies on the mutagenicity of tris(2,3-dibromopropyl) phosphate (tris-BP) and its metabolites in salmonella

A series of methylated metabolites of the flame retardant tris(2,3-dibromopropyl) phosphate (Tris-BP) were tested for mutagenicity in Salmonella, along with the parent chemical and other structurally related chemicals. The metabolites produced a gradient of mutagenic responses in TA1535 in the same dose range and up to the same magnitude as the Tris-BP response. None of the metabolites tested appeared to be the ultimate mutagen, since they all required S-9 for their mutagenic activity.     

Di‐(2‐ethylhexyl) phthalate and bisphenol A exposure impairs mouse primordial follicle assembly in vitro

Bisphenol‐A (BPA) and diethylhexyl phthalate (DEHP) are estrogenic compounds widely used in commercial plastic products. Previous studies have shown that exposure to such compounds have adverse effects on various aspects of mammalian reproduction including folliculogenesis. The objective of this study was to examine the effects of BPA and DEHP exposure on primordial follicle formation. We found that germ cell nest breakdown and primordial follicle assembly were significantly reduced when newborn mouse ovaries were exposed to 10 or 100 μM BPA and DEHP in vitro. Moreover, BPA and DEHP exposure increased the number of TUNEL positive oocytes and the mRNA level of the pro‐apoptotic gene Bax in oocytes. These effects were associated with decreased expression of oocyte specific genes such as LIM homeobox 8 (Lhx8), factor in the germline alpha (Figla), spermatogenesis and oogenesis helix‐loop‐helix (Sohlh2), and newborn ovary homeobox (Nobox). Interestingly, BPA and DEHP exposure also prevented DNA demethylation of CpG sites of the Lhx8 gene in oocytes, a process normally associated with folliculogenesis. Finally, folliculogenesis was severely impaired in BPA and DEHP exposed ovaries after transplantation into the kidney capsules of immunodeficient mice. In conclusion, BPA and DEHP exposures impair mouse primordial follicle assembly in vitro. Environ. Mol. Mutagen. 55:343–353, 2014. © 2014 Wiley Periodicals, Inc.     

Biodegradation of bis(2-hydroxyethyl) terephthalate by a newly isolated Enterobacter sp. HY1 and characterization of its esterase properties

Bis(2-hydroxyethyl) terephthalate (BHET) is an important compound produced from poly(ethylene terephthalate) (PET) cleavage. It was selected as the representative substance for the study of PET degradation. A bacterial strain HY1 that could degrade BHET was isolated and identified as Enterobacter sp. The optimal temperature and pH for BHET biodegradation were determined to be 30°C and 8.0, respectively. The half-life of degradation was 70.20 h at an initial BHET concentration of 1,000 mg/L. The results of metabolites' analysis by liquid chromatograph–mass spectrometer revealed that BHET was first converted to mono-(2-hydroxyethyl) terephthalate (MHET) and then to terephthalic acid. Furthermore, an esterase-encoding gene, estB, was cloned from strain HY1, and the expressed enzyme EstB was characterized. The esterase has a molecular mass of approximately 25.13 kDa, with an isoelectric point of 4.68. Its optimal pH and temperature were pH 8.0 and 40°C, respectively. The analysis of the enzymatic products showed that EstB could hydrolyze one ester bond of BHET to MHET. To the best of authors' knowledge, this is the first report on the biodegradation characteristics of BHET by a member of the Enterobacter genus.     

Mutagenicity and toxicity studies of several α,β-unsaturated aldehydes in the Salmonella typhimurium mutagenicity assay

α,β-Unsaturated aldehydes are reactive compounds which are ubiquitous in the environment. This class of compounds has been tested for mutagenicity in Salmonella typhimurium by a number of groups who have obtained differing results. The present studies were undertaken to test the mutagenicity and toxicity of two novel α,β-unsaturated aldehydes, specifically trans,trans-muconaldehyde and trans-4-hydroxynonenal, and to re-examine the mutagenicity of crotonaldehyde. Trans,trans-muconaldehyde is a newly found microsomal metabolite of benzene, and trans-4-hydroxynonenal is a toxic aldehyde formed endogenously during lipid peroxidation. Compounds were tested in S. typhimurium strain TA 100 using a 30-min liquid preincubation procedure. The present mutagenicity studies indicate that these α,β-unsaturated aldehydes at first appear to be mutagenic, although only at concentrations which decrease survival counts, and result in a disappearance of the bacterial lawn. The colonies observed on mutagenicity test plates are not mutants but rather pin point survivors.