Di-n-butyl phthalate induces toxicity in male fetal mouse testicular development by regulating the MAPK signaling pathway.

"White pollution" has a significant impact on male reproduction. Di-n-butyl phthalate (DBP) is one of the most important factors in this type of pollution. Currently, research from international sources has demonstrated the significant reproductive toxicity of DBP. However, most of these studies have focused mainly on hormones expression at the protein and mRNA levels and the specific molecular targets of DBP and its mechanisms of action remain unclear. In this study, we established a Sprague Dawley pregnant mouse model exposed to DBP, and all male offspring were immediately euthanized at birth and bilateral testes were collected. We found through transcriptome sequencing that cell apoptosis and MAPK signaling pathway are the main potential pathways for DBP induced reproductive toxicity. Molecular biology analyses revealed a significant increase in the protein levels of JNK1(MAPK8) and BAX, as well as a significant increase in the BAX/BCL2 ratio after DBP exposure. Therefore, we propose that DBP induces reproductive toxicity by regulating JNK1 expression to activate the MAPK signaling pathway and induce reproductive cell apoptosis. In conclusion, our study provides the first evidence that the MAPK signaling pathway is involved in DBP-induced reproductive toxicity and highlights the importance of JNK1 as a potential target of DBP in inducing reproductive toxicity.

Biodegradation of phthalate acid esters and whole-genome analysis of a novel Streptomyces sp. FZ201 isolated from natural habitats.

Di-n-butyl phthalate (DBP) is one of the most extensively used phthalic acid esters (PAEs) and is considered to be an emerging, globally concerning pollutant. The genus Streptomyces holds promise as a degrader of various organic pollutants, but PAE biodegradation mechanisms by Streptomyces species remain unsolved. In this study, a novel PAE-degrading Streptomyces sp. FZ201 isolated from natural habitats efficiently degraded various PAEs. FZ201 had strong resilience against DBP and exhibited immediate degradation, with kinetics adhering to a first-order model. The comprehensive biodegradation of DBP involves de-esterification, ß-oxidation, trans-esterification, and aromatic ring cleavage. FZ201 contains numerous catabolic genes that potentially facilitate PAE biodegradation. The DBP metabolic pathway was reconstructed by genome annotation and intermediate identification. Streptomyces species have an open pangenome with substantial genome expansion events during the evolutionary process, enabling extensive genetic diversity and highly plastic genomes within the Streptomyces genus. FZ201 had a diverse array of highly expressed genes associated with the degradation of PAEs, potentially contributing significantly to its adaptive advantage and efficiency of PAE degradation. Thus, FZ201 is a promising candidate for remediating highly PAE-contaminated environments. These findings enhance our preliminary understanding of the molecular mechanisms employed by Streptomyces for the removal of PAEs.

Complete degradation of di-n-butyl phthalate by Glutamicibacter sp. strain 0426 with a novel pathway.

Di-n-butyl phthalate (DBP) is widely used as plasticizer that has potential carcinogenic, teratogenic, and endocrine effects. In the present study, an efficient DBP-degrading bacterial strain 0426 was isolated and identified as a Glutamicibacter sp. strain 0426. It can utilize DBP as the sole source of carbon and energy and completely degraded 300 mg/L of DBP within 12 h. The optimal conditions (pH 6.9 and 31.7 °C) for DBP degradation were determined by response surface methodology and DBP degradation well fitted with the first-order kinetics. Bioaugmentation of contaminated soil with strain 0426 enhanced DBP (1 mg/g soil) degradation, indicating the application potential of strain 0426 for environment DBP removal. Strain 0426 harbors a distinctive DBP hydrolysis mechanism with two parallel benzoate metabolic pathways, which may account for the remarkable performance of DBP degradation. Sequences alignment has shown that an alpha/beta fold hydrolase (WP_083586847.1) contained a conserved catalytic triad and pentapeptide motif (GX1SX2G), of which function is similar to phthalic acid ester (PAEs) hydrolases and lipases that can efficiently catalyze hydrolysis of water-insoluble substrates. Furthermore, phthalic acid was converted to benzoate by decarboxylation, which entered into two different pathways: one is the protocatechuic acid pathway under the role of pca cluster, and the other is the catechol pathway. This study demonstrates a novel DBP degradation pathway, which broadens our understanding of the mechanisms of PAE biodegradation.

Cytoprotective potency of naringin against di-n-butylphthalate (DBP)-induced oxidative testicular damage in male rats.

The present study aimed to investigate the protective potential of naringin (NG) against di-n-butyl phthalate (DBP)- induced testicular damage and impairment of spermatogenesis in rats. Forty-two male Wistar albino rats were divided into six equal groups, and treated orally, 3 times weekly for 8 successive weeks. Control vehicle group was administrated olive oil, naringin-treated group was administered NG (80 mg/kg), DBP 250- and DBP 500- intoxicated groups received DBP (250 mg/kg) and (500 mg/kg), respectively, NG + DBP 250 and NG + DBP 500 groups received NG, an hour prior to DBP 250 and 500 administration. The results revealed that DBP induced dose-dependent male reproductive dysfunctions, included a significant decrease in the serum testosterone level concomitantly with significant decreases in the sperm count, viability, and total motility. Meanwhile, DBP significantly increased the testicular malondialdehyde level with significant reductions of glutathione content and catalase activity. Histopathologically, DBP provoked absence of spermatozoa, degenerative changes in the cell layers of seminiferous tubules and a significant decrease in the thickness of the seminiferous tubules epithelium. Conversely, the concomitant treatment with NG, one hour before DBP 250 or 500- intoxication mitigated the dose-dependent reproductive dysfunctions induced by DBP, evidenced by significant increases of serum testosterone level, sperm motility, count and viability along with marked improvement of the oxidant/antioxidant status and testicular histoarchitecture. In conclusion, the findings recorded herein proved that NG could mitigate DBP-induced testicular damage and impairment of spermatogenesis, suggesting the perspective of using NG as a natural protective and therapeutic agent for alleviating the reproductive dysfunctions and improving reproductive performance, mainly via its potent antioxidant activity.

Curcumin strengthens a spontaneous self-protective mechanism-SP1/PRDX6 pathway, against di-n-butyl phthalate-induced testicular ferroptosis damage.

As one of the common plasticizers, di-n-butyl phthalate (DBP) has been using in various daily consumer products worldwide. Since it is easily released from products and exists in the environment for a long time, it has a lasting impact on human health, especially male reproductive health. However, the detailed mechanism of testicular damage from DBP and the protection strategy are still not clear enough. In this study, we found that DBP could induce dose-dependent ferroptosis in testicular tissue. Mechanism dissection indicates that DBP can upregulate SP1 expression, which could directly transcriptionally upregulate PRDX6, a negative regulator of ferroptosis. Overexpression of PRDX6 or adding SP1 agonist curcumin could suppress the DBP-induced ferroptosis on testicular cells. In vivo, rats were given 500 mg/kg/day DBP orally for 3 weeks; elevated levels of ferroptosis were detected in testicular tissue. When the above-mentioned doses of DBP and curcumin at a dose of 300 mg/kg/day were administered intragastrically simultaneously, the testicular ferroptosis induced by DBP was alleviated. Immunohistochemistry and quantitative real-time PCR of testis tissue showed that the expression of PRDX6 was upregulated under the action of DBP and curcumin. These findings suggest a spontaneous self-protection mechanism of testicular tissue from DBP damage by upregulating SP1 and PRDX6. However, it is not strong enough to resist the DBP-induced ferroptosis. Curcumin can strengthen this self-protection mechanism and weaken the level of ferroptosis induced by DBP. This study may help us to develop a novel therapeutic option with curcumin to protect the testicular tissue from ferroptosis and function impairment by DBP.

The no-observed-adverse-effect level of phthalates promotes proliferation and cell cycle progression in normal human breast cells.

OBJECTIVE: The data on the association between phthalates and breast cancer risk remains inconsistent. This study aimed to explore the possible mechanism of low-dose exposures of phthalates, including Butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(20ethylhexyl) phthalate (DEHP), on breast tumorigenesis. METHODS AND METHODS: MCF-10A normal breast cells were treated with phthalates (10 and 100 nM) and 17ß-estradiol (E2, 10 nM), which were co-cultured with fibroblasts from normal mammary tissue. Cell viability, cycle, and apoptosis were detected by MTT assay, flow cytometry, and TUNEL assay respectively. The expression levels of related proteins were determined by Western blot. RESULTS: Like E2, both 10 nM and 100 nM phthalates exerted significantly higher cell viability, lower apoptosis, and increased cell numbers in the S and G2/M phases with up-regulation of cyclin D/CDK4, cyclin E/CDK2, cyclin A/CDK2, cyclin A/CDK1, and cyclin B/CDK1, compared with the control group. Significant increase in PDK1, P13K, p-AKT, p-mTOR, and BCL-2 expression and a decrease in Bax protein, cytochrome C, caspase 8, and caspase 3 levels were noted in cells treated with 10 nM and 100 nM phthalates and E2, compared with the control group and MCF-10A cells co-cultured with fibroblasts. The effects of the three phthalates were noted to be dose-dependent. CONCLUSIONS: The results indicate that phthalates at a level below its no-observed-adverse-effect concentration, as defined by the current standards, still induce cell cycle progression and proliferation as well as inhibit apoptosis of normal breast cells. Thus, the possibility of breast tumorigenesis through chronic phthalate exposure should be considered.

Effects of Dibutylphthalate and Steroid Hormone Mixture on Human Prostate Cells.

Phthalates are a family of aromatic chemical compounds mainly used as plasticizers. Among phthalates, di-n-butyl phthalate (DBP) is a low-molecular-weight phthalate used as a component of many cosmetic products, such as nail polish, and other perfumed personal care products. DBP has toxic effects on reproductive health, inducing testicular damage and developmental malformations. Inside the male reproductive system, the prostate gland reacts to both male and female sex steroids. For this reason, it represents an important target of endocrine-disrupting chemicals (EDCs), compounds that are able to affect the estrogen and androgen signaling pathways, thus interfering with prostate homeostasis and inducing several prostate pathologies. The aim of this project was to investigate the effects of DBP, alone and in combination with testosterone (T), 17ß-estradiol (E2), and both, on the normal PNT1A human prostate cell-derived cell line, to mimic environmental contamination. We showed that DBP and all of the tested mixtures increase cell viability through activation of both estrogen receptor α (ERα) and androgen receptor (AR). DBP modulated steroid receptor levels in a nonmonotonic way, and differently to endogenous hormones. In addition, DBP translocated ERα to the nucleus over different durations and for a more prolonged time than E2, altering the normal responsiveness of prostate cells. However, DBP alone seemed not to influence AR localization, but AR was continuously and persistently activated when DBP was used in combination. Our results show that DBP alone, and in mixture, alters redox homeostasis in prostate cells, leading to a greater increase in cell oxidative susceptibility. In addition, we also demonstrate that DBP increases the migratory potential of PNT1A cells. In conclusion, our findings demonstrate that DBP, alone and in mixtures with endogenous steroid hormones, acts as an EDC, resulting in an altered prostate cell physiology and making these cells more prone to cancer transformation.

DBP exposure induces thyroid inflammatory impairment through activating AKT/NF-κB/NLRP3 signaling.

Previous studies exhibited reproductive and neurodevelopmental toxicity in rats exposed to Di-n-butyl phthalate (DBP). However, the effects of DBP exposure on the other endocrine organ are still unclear. This study aimed to assess the impact of DBP exposure on the thyroid of male rats and the associated mechanisms. Here, rats were respectively treated with DBP at 0 (control), 50 (low dose), 250 (medium dose), or 500 (high dose) mg/kg/day dissolved in 1 ml quantity of corn oil by intragastrical administration for two weeks. The results demonstrated that the proliferation and inflammatory response changes were significantly different compared to the control. In vivo DBP is mainly converted to mono-n-butyl phthalate (MBP), an active form producing untoward reactions of DBP exposure. Therefore, for in vitro experiments, we treated the thyroid follicular epithelial cell line (Nthy-ori 3-1) in a temporal gradient using 1 mM MBP. Further in vitro studies showed that MBP exposure upregulated tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), as well as interleukin-1ß (IL-1ß) by activating AKT/NF-κB/NLRP3 signaling. Meanwhile, we detected that Pellino2 (Peli2) played an essential role in promoting the activation of NLRP3 inflammasome. Briefly speaking, this study confirmed that DBP exposure caused impaired thyroid structure and thyroid inflammation in male rats, which offered new views into the harm of DBP exposure on the endocrine organ.

Interaction between Phthalate Ester and Rice Plants: Novel Transformation Pathways and Metabolic-Network Perturbations.

Our understanding is limited concerning the interaction mechanism between widespread phthalate esters and staple crops, which have strong implications for human exposure. Therefore, this study was aimed at illuminating the transformation pathways of di-n-butyl phthalate (DnBP) in rice using an untargeted screening method. UPLC-QTOF-MS identified 16 intermediate transformation products formed through hydroxylation, hydrolysis, and oxidation in phase I metabolism and further by conjugation with amino acids, glutathione, and carbohydrates in phase II metabolism. Mono-2-hydroxy-n-butyl phthalate-l-aspartic acid (MHBP-asp) and mono-2-hydroxy-n-butyl phthalate-d-alanyl-ß-d-glucoside (MHBP-ala-glu) products were observed for the first time. The proteomic analysis demonstrated that DnBP upregulated the expression of rice proteins associated with transporter activity, antioxidant synthesis, and oxidative stress response and downregulated that of proteins involved in photosynthesis, photorespiration, chlorophyll binding, and mono-oxygenase activity. Molecular docking revealed that DnBP can affect protein molecular activity via pi-sigma, pi-alkyl, and pi-pi interactions or by forming carbon-hydrogen bonds. The metabolomic analysis showed that key metabolic pathways including citrate cycle, biosynthesis of aminoacyl-tRNA, and metabolism of amino acids, sphingolipids, carbohydrates, nucleotides, and glutathione were activated in rice plants exposed to DnBP and its primary metabolite mono-n-butyl phthalate (MnBP). Furthermore, exposure to 80 ng/mL MnBP significantly perturbed the metabolic profile and molecular function in plants, with downregulation of the levels of beta-alanine (0.56-fold), cytosine (0.48-fold), thymine (0.62-fold), uracil (0.48-fold), glucose (0.59-fold), and glucose-1-phosphate (0.33-fold), as well as upregulation of the levels of l-glutamic acid (2.97-fold), l-cystine (2.69-fold), and phytosphingosine (38.38-fold). Therefore, the degradation intermediates of DnBP pose a potentially risk to plant metabolism and raise concerns for crop safety related to plasticizer pollution.

Regulation of the cell cycle and P13K/AKT/mTOR signaling pathway by phthalates in normal human breast cells.

OBJECTIVE: To investigate the impact of phthalates, including Butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP), in breast carcinogenesis. MATERIALS AND METHODS: MCF-10A normal breast cells were treated with phthalates (100 nM) and 17ß-estradiol (E2, 10 nM), which were co-cultured with fibroblasts from normal mammary tissue adjacent to estrogen receptor positive primary breast cancers. Cell viability was determined using a 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycles were analyzed using flow cytometry. The proteins involving cell cycles and P13K/AKT/mTOR signaling pathway were then evaluated by Western blot analysis. RESULTS: MCF-10A co-cultured cells treated with E2, BBP, DBP, and DEHP exhibited a significant increase in cell viability using MTT assay. The expressions of P13K, p-AKT, and p-mTOR, as well as PDK1 expression, were significantly higher in MCF-10A cells treated with E2 and phthalates. E2, BBP, DBP, and DEHP significantly increased cell percentages in the S and G2/M phases. The significantly higher expression of cyclin D/CDK4, cyclin E/CDK2, cyclin A/CDK2, cyclin A/CDK1, and cyclin B/CDK1 in MCF-10A co-cultured cells were induced by E2 and these three phthalates. CONCLUSION: These results provide consistent data regarding the potential role of phthalates exposure in the stimulating proliferation of normal breast cells, enhancing cell viability, and driving P13K/AKT/mTOR signaling pathway and cell cycle progression. These findings strongly support the hypothesis that phthalates may play a crucial role in breast tumorigenesis.

Di-n-butyl phthalate stress hampers compost multifunctionality by reducing microbial biomass, diversity and network complexity.

Phthalates are common pollutants in agriculture. Here, the influence of di-n-butyl phthalate (DBP) on multifunctionality of composting was assessed. Results indicated that DBP stress (100 mg/kg) hampered multifunctionality from the thermophilic phase onwards and resulted in a 6.5 % reduction of all assessed functions. DBP stress also significantly reduced microbial biomass (P < 0.05), altered microbial composition (P < 0.05), and decreased network complexity (P < 0.01). Multifunctionality was found to be strongly correlated (P < 0.001) with microbial biomass, diversity, and network complexity. In addition, keystone taxa responsive to DBP were identified as Streptomyces, Thermoactinomyces, Mycothermus, and Lutispora. These taxa were significantly (P < 0.001) affected by DBP stress, and a correlation between them and multifunctionality was shown. This study contributes to a better understanding of the negative implications of phthalates during composting processes, which is of great significance to the development of new treatment strategies for agricultural waste.

Changes in testicular histomorphometry and ultrastructure of Leydig cells in adult male Japanese quail exposed to di (n-butyl) phthalate (DBP) during the prepubertal period.

Phthalate esters, such as di(n-butyl) phthalate, (DBP), are synthetic chemical pollutants commonly used as plasticizers in the manufacture of plastics. In the present study, we investigated the effects of DBP in the testes of adult male quails (Coturnix cortunix japonica) exposed by oral gavage to variable doses of DBP (0 [control], 1, 10, 50, 200, and 400 mg/kgbw-d), for 30 days during the prepubertal period, using histo-morphometric and ultrastructural techniques. Generally, significant decreases in seminiferous tubular diameter (STD) and epithelial height (SEH) were observed predominantly at the highest DBP doses (200 and 400 mg/kg), as compared to medium (50 mg/kg), and lowest doses (1 and 10 mg/kg) as well as the control group. Ultrastructurally, apparent dose-specific degenerative changes were observed in the Leydig cells. The lowest DBP doses (1 and 10 mg/kg) did not produce significant effects on Leydig cell ultrastructure, whereas, at the highest doses (200 and 400 mg/kg), the Leydig cells were remarkably conspicuous in the interstitium and appeared foamy. There was a preponderance of electron-lucent lipid droplets which crowded out the normal organelles of the cell, as well as increases in the number of dense bodies in the cytoplasm. The smooth endoplasmic reticulum (sER) was less obvious, compacted, and wedged between the abundant lipid droplets and mitochondria. Taken together, these findings indicate that pre-pubertal exposure of precocious quail birds to DBP, produced parameter-specific histometric tubular changes, as well as dose-dependent cyto-structural derangement of the Leydig cells; which consequently may lead to overt reproductive impairments in the adult bird in the environment.

Role of ROS/JAK2/STAT3 signaling pathway in di-n-butyl phthalate-induced testosterone synthesis inhibition and antagonism of lycopene.

Di-n-butyl phthalate (DBP) causes adverse effects on male reproduction, especially testosterone synthesis inhibition. However, the specific mechanism of DBP-induced testosterone synthesis inhibition and its effective intervention measures of prevention and treatment are scarce presently. Lycopene (LYC) plays beneficial roles in male infertility because of its antioxidant activity. Nevertheless, it is unclear whether LYC could prevent DBP-induced male reproductive toxicity. By in vitro and in vivo investigations, this study demonstrated that DBP activated ROS/JAK2/STAT3 signaling pathway, promoted mitophagy and apoptosis, which in turn inhibited testosterone synthesis. Additionally, another major finding was that LYC supplement could reverse the above change, presenting as the restraint of ROS/JAK2/STAT3 signaling pathway, reduction of mitophagy and apoptosis, and improvement of testosterone synthesis. Our study facilitates deeper understandings of the mechanism in DBP-induced testosterone synthesis inhibition, and identifies LYC as the effective prevention and control strategies for DBP poisoning.

Effect of di(n-butyl) phthalate on the blood-testis barrier during puberty onset.

Di(n-butyl) phthalate (DBP) is considered a substance of serious concern because of its reproductive toxicity and endocrine-disrupting properties. Exposure to DBP causes morphological and functional changes in the male reproductive system of birds and mammals. However, there are no detailed reports on the effects of DBP on the Sertoli cell and junctional complexes of the blood-testis barrier (BTB) in birds. The present study investigated dose-related ultrastructural changes in Sertoli cells and junctional complexes of the BTB in adult Japanese quail (Coturnix coturnix japonica) exposed to DBP prior to puberty. A total of 25 Japanese quail were used for the study. Exposure to DBP doses of 50, 200 and 400 mg DBP/kg/d caused dose-related ultrastructural changes in junctional complexes including dilation and separation, while disruption of cytoplasmic membranes and mitochondria was observed in Sertoli cells. There was a significant difference in the sum of vacuoles, vacuole diameter, nuclear width, nuclear length, nuclear area, sum of damaged spherical mitochondria, width of elongated mitochondria and the sum of damaged elongated mitochondria among the five treatment groups (p Ë‚ 0.05). Prepubertal exposure to DBP at doses of 50, 200 and 400 mg DBP/kg/d for 30 days led to adverse effects in the adult male Japanese quail reproductive system by inducing structural changes in the Sertoli cells and junctional complexes. Such changes might disrupt the BTB and potentially interfere with spermatogenesis. Results indicated that the Sertoli cell is sensitive to DBP exposure and might be an important cellular target for DBP-induced testicular toxicity.

Prenatal and perinatal phthalate exposure is associated with sex-dependent changes in hippocampal miR-15b-5p and miR-34a-5p expression and changes in testicular morphology in rat offspring.

MicroRNAs are a large group of non-coding nucleic acids, usually 20-22 nt long, which bind to regulatory sections of messenger RNA (mRNA) and inhibit gene expression. However, genome activity is also regulated by hormones. Endocrine disruptors such as those from the phthalate group imitate or block these hormonal effects, and our previous study showed a long-lasting decrease in plasma testosterone levels in rat offspring exposed to a mixture of three phthalates in utero and postnatally. These effects were also observed at the behavioural level. To shed more light on these findings, in this new study we compared testicular tissue morphology between control and phthalatetreated males and investigated possible persistent changes and sex differences in the expression of two hippocampal microRNAs - miR- 15b-5p and miR-34a-5p - participating in the transcription of steroidogenic genes. Histologically observed changes in testicular tissue morphology of phthalate-exposed males compared to control support testosterone drop observed in the previous study. At the microRNA level, we observed more significant changes in phthalate-treated females than in males. However, we are unable to relate these effects to the previously observed behavioural changes.

Phthalate plasticizer decreases the prion-like protein doppel essential for structural integrity and function of spermatozoa.

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.

Using fluorescence and circular dichroism (CD) spectroscopy to investigate the interaction between di-n-butyl phthalate and bovine serum albumin.

The interaction between di-n-butyl phthalate (DBP) and bovine serum albumin (BSA) in physiological Tris-HCl buffer at pH 7.4 was investigated by fluorescence quenching technique. By analyzing the fluorescence spectrum and intensity, it was observed that the DBP had a strong ability to quench the intrinsic fluorescence of BSA through a static quenching procedure. The binding constants K and the number of binding sites n of DBP with BSA were calculated to be 0.11 × 102 L·mol-1 and 0.52 at 298 K, respectively. The thermodynamic parameters of enthalpy change (ΔH) and entropy change (ΔS) were also calculated to be positive showing that hydrophobic forces might play a major role in the binding of DBP to BSA. The binding process was spontaneous in which Gibbs free energy change (ΔG) was negative. The distance (r) between the donor (BSA) and acceptor (DBP) was calculated to be 2.02 nm based on Forster's non-radiative energy transfer theory, which indicated that the energy transfer from BSA to DBP occurs with a high possibility. The synchronous fluorescence, three-dimensional fluorescence, and circular dichroism (CD) spectra showed that the binding of di-n-butyl phthalate to BSA induced conformational changes in BSA. The interaction between DBP and BSA can help researchers better understand the nature of poisons and serve people in the right way with first aid and detoxification.

Evaluation of di-n-butyl phthalate reproductive toxicity in pregnant rats and their offspring and assessment of vitamin E administration in reducing toxicity.

Phthalates are environmental contaminants mostly used as plasticizers and additives in different products. Having endocrine-disrupting properties, phthalates are known as potential reproductive toxicants. The present study was conducted to evaluate the reproductive toxicity of di-n-butyl phthalate (DBP) in pregnant rats and their offspring and also to assess the ability of vitamin E in the elimination or reducing reproductive toxicity of DBP. Sixty-six pregnant Wistar rats were exposed to 100, 500 or 1,000 mg kg-1 per day DBP or 500 mg kg-1 per day DBP along with 100 mg kg-1 per day vitamin E during gestation. After delivery, they were divided into two groups. In one group gavage was finished after litter while in the other DBP administration was continued till weaning. The results showed that DBP affected many aspects of reproductive performance in pregnant rats and their offspring. It could be suggested that vitamin E could ameliorate the adverse effects of DBP, especially in male pups.

Characteristics and Health Risks of Phthalate Ester Contamination in Soil and Plants in Coastal Areas of South China.

Phthalate esters (PAEs) are widely used as plasticizers in industrial and commercial products, and are classified as endocrine-disrupting compounds. In this study, we investigated the contamination characteristics and health risks of PAEs in the soil-plant system in coastal areas of South China. PAEs were detected in soil and plant samples at all 37 sampling sites. The total concentration of the 15 PAEs in soil samples ranged from 0.445 to 4.437 mg/kg, and the mean concentration was 1.582 ± 0.937 mg/kg. The total concentration of the 15 PAEs in plant samples ranged from 2.176 to 30.276 mg/kg, and the mean concentration was 8.712 ± 5.840 mg/kg. Di(2-Ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DnBP) were the major PAEs compounds in all samples. The selected contaminants exhibited completely different spatial distributions within the study area. Notably, higher concentrations of PAEs were found in the coastal Guangdong Province of South China. The average noncarcinogenic risks of Σ6 PAEs were at acceptable levels via dietary and nondietary routes. However, the noncarcinogenic risks posed by DEHP and DBP at some sampling sites were relatively high. Furthermore, dietary and nondietary carcinogenic risks were very low for BBP, but carcinogenic risks posed by DEHP via diet. The results suggest that PAEs in the coastal soil-plant system in South China, through human risk assessment, will induce some adverse effects on human health, especially in children. This study provides an important basis for risk management of PAEs in agriculture, and safety in coastal areas of South China.

Di-n-butyl phthalate regulates vascular smooth muscle cells phenotypic switching by MiR-139-5p-MYOCD pathways.

Di-n-butyl phthalate (DBP) is ubiquitous in environment and has been detected in almost all human bodies. Few data could be found about the effects of DBP on cardiovascular system, though its reproductive toxicities have been studied extensively. This study aimed to explore effects of DBP on phenotypic switching of vascular smooth muscle cells (VSMCs), an essential step during the formation of atherosclerosis (AS). A7r5 cells were employed and exposed to various levels of DBP (10-9, 10-8, 10-7, 10-6, and 10-5 M) or DMSO as control. CCK-8 assay was used to detect the effects of DBP on cell viability. Expressions of mRNA/miRNAs and proteins were measured by qRT-PCR and western blotting, respectively. Bioinformatic analysis and dual-luciferase reporter assay were used to analyze the combination between miR-139-5p and Myocardin (MYOCD). Results revealed that DBP at 10-7 M prompted phenotypic switching from contractile to synthetic of VSMCs by inhibiting contractile VSMCs marker genes via suppressing the expression of MYOCD. Moreover, miR-139c-5p directly targeted MYOCD 3'UTR and modulated MYOCD expression. Besides, DBP inhibited the expression of MYOCD and VSMCs marker genes by upregulating miR-139-5p. Collectively, these data suggested that DBP could promote the phenotypic switching from contractile to synthetic of VSMCs in A7r5 cells through miR-139-5p-MYOCD.