Nonylphenol Isomers Research Articles

Nontarget and suspect screening reveals the presence of multiple plastic-related compounds in polar bear, killer whale, narwhal and long-finned pilot whale blubber from East Greenland

The monitoring of legacy contaminants in sentinel northern marine mammals has revealed some of the highest concentrations globally. However, investigations into the presence of chemicals of emerging Arctic concern (CEACs) and other lesser-known chemicals are rarely conducted, if at all. Here, we used a nontarget/suspect approach to screen for thousands of different chemicals, including many CEACs and plastic-related compounds (PRCs) in blubber/adipose from killer whales (Orcinus orca), narwhals (Monodon monoceros), long-finned pilot whales (Globicephala melas), and polar bears (Ursus maritimus) in East Greenland. 138 compounds were tentatively identified mostly as PRCs, and four were confirmed using authentic standards: di(2-ethylhexyl) phthalate (DEHP), diethyl phthalate (DEP), di(2-propylheptyl) phthalate (DPHP), and one antioxidant (Irganox 1010). Three other PRCs, a nonylphenol isomer, 2,6-di-tert-butylphenol, and dioctyl sebacate, exhibited fragmentation patterns matching those in library databases. While phthalates were only above detection limits in some polar bear and narwhal, Irganox 1010, nonylphenol, and 2,6-di-tert-butylphenol were detected in >50% of all samples. This study represents the first application of a nontarget/suspect screening approach in Arctic cetaceans, leading to the identification of multiple PRCs in their blubber. Further nontarget analyses are warranted to comprehensively characterize the extent of CEAC and PRC contamination within Arctic marine food webs.

Filling the Blank Space: Branched 4-Nonylphenol Isomers Are Responsible for Robust Constitutive Androstane Receptor (CAR) Activation by Nonylphenol.

4-Nonylphenol (4-NP), a para-substituted phenolic compound with a straight or branched carbon chain, is a ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor (ED) with potent activities on estrogen receptors. Constitutive Androstane Receptor (CAR) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in the ED mechanism of action. An NP mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by 4-NP are enigmatic, and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here, we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR variants using molecular dynamics (MD) simulations, cellular experiments with various CAR expression constructs, recombinant CAR LBD in a TR-FRET assay, or a differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type CAR1 and CAR3 variant LBDs in MD simulations. Consistently, branched 4-NPs activated CAR3 and CAR1 LBD more efficiently than linear 4-NP. Furthermore, in HepaRG cells, we observed that all 4-NPs upregulated CYP2B6 mRNA, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and human CAR-LBD, as well as its dominant variant CAR3. The work could contribute to the safer use of individual 4-NPs in many areas of industry.

The degradation of a nonylphenol isomer in water and soil of typical sewage irrigation area in China

AbstractNonylphenols (NPs), one of the known endocrine disrupting organic pollutants, had received full attention for their estrogenic potency and ubiquitous distribution in the environment. Due to its chemical properties and compositions of technical nonylphenols (tNPs), which was persistent and soil environments for a long time and affect the ecosystem vigorously, degradation behaviour of a nonylphenol isomer (NP38) and the para‐substituded nonylphenols (4‐NPs) in water and soil was compared in this study. The results showed that the 4‐NPs and NP38 could be degraded under the mercury lamp in ultrapure water, 90% of 4‐NPs and NP38 were photodegradated in 2 h, but the removal rate of the 4‐NPs was slightly faster than the NP38. The NP38 was almost completely decay under the UV in 120 min. The active species (NO3− and Fe3+) were benefit for the photogradation of NP38 and presented higher efficiency under the higher concentration. The biodegradable curves of the 4‐NPs and NP38 in the SS (The soil from sewage irrigation area) were similar, 84% of the NP38 was removed after 20 days later in SS, the 62.25% of the NP38 was remained after 60 days later in CS (the soil was from the groundwater irrigation area), but the removal rate of the NP38 was nearly two times to the 4‐NPs in the CS, and the structure of the nonylphenol isomers with α‐quaternary had influenced on its degradation in soils. The study was helpful for better understanding the migration and transformation behaviour of NPs and provided scientific basis for accurate assessment of environmental risk of NPs from the sewage irrigation area.

New findings on the occurrence, removal, and risk assessment of nonylphenol and octylphenol in industrial wastewater treatment plants in Korea

Nonylphenol (NP) and octylphenol (OP), extensively used in industries, can disrupt the human endocrine system and cause significant ecological toxicity. Therefore, in this study, we aimed to reveal the occurrence and removal characteristics of NP and OP in 30 industrial wastewater treatment plants (IWWTPs). Specifically, this study focused on 13 NP isomers that have not been previously reported. Additionally, the potential environmental risk of NP and OP discharged from IWWTPs was assessed using a minimum dilution factor (MDF), proposed for the first time in this study. We showed that the concentration and proportion of the isomer NP11 were higher than those of the other isomers in the IWWTP influents and effluents. We also identified an activated sludge-activated carbon adsorption process suitable for removing NP and OP. Finally, we proposed the MDF value of 11 for the potential environmental risk assessment of NP and OP, revealing that OP poses a higher risk than NP when discharged into surface water. These findings underscore the importance of focusing on the isomer NP11 and OP in IWWTPs.

Efficient Removal of Nonylphenol Isomers from Water by Use of Organo-Hydrotalcites.

The presence of potent organic endocrine-disrupting chemicals (EDCs) in natural aquifers can have adverse impacts on public health and the environment. 4-nonylphenol, one such EDC, can be efficiently removed from water by adsorption onto a clayey material. In this work, we created an effective sorbent for this purpose by using co-precipitation and subsequent ion-exchange to intercalate the organic anion deoxycholate into a Mg/Al hydrotalcite. Intercalating deoxycholate ions increased the organophilicity of the hydrotalcite surface. The solid was used to adsorb 4-nonylphenol at different pollutant concentrations and temperatures. The adsorption process was subjected to a kinetic study. Based on the results, the EDC was adsorbed by chemisorption. In addition, based on the equilibrium isotherms used for the process, the Freundlich model was the most accurate in reproducing the adsorption of 4-nonylphenol onto deoxycholate-intercalated hydrotalcite.

Structure–Biodegradability Relationship of Nonylphenol Isomers in Two Soils with Long-Term Reclaimed Water Irrigation

Nonylphenol (NP), as one of the typical endocrine disrupter chemicals (EDCs), has a high detection concentration and frequency in reclaimed water. This research focused on the degradation of NP isomers in two typical reclaimed water irrigation fields in Daxing, China, and Florida, USA. The results showed that the half-lives of NP isomer degradation in the soil of China and Florida were 2.03–8.66 d and 5.16–11.83 d, respectively. The degradation of NP isomers was structure-specific. Isomers of NP5, NP2, NP11, and NP3 had the highest degradation rates in the two soils; NP12, NP7, and NP6 were the isomers with medium degradation rates; and NP4, NP1, NP10, NP9, and NP8 had the slowest degradation rates. Steric hindrance and mean information index for the magnitude of distance (IDWbar) were found to be the better indexes for measuring the degradation of NP isomers compared with the length of the side chain, the type of the substitute, and the molecular connectivity. This study offers insights into the characteristics of NP isomers and two reliable indicators for measuring the degradation of NP isomers, which could provide data support for the environmental fate and the health risk assessment of NP in the future.

4-[1-Ethyl-1-methylhexy]-phenol induces apoptosis and interrupts Ca2+ homeostasis via ROS pathway in Sertoli TM4 cells.

Biological effect of an individual nonylphenol (NP) isomer extremely relies upon the side chain structure. This research was designed to evaluate the impact of NP isomer, 4-[1-ethyl-1-methylhexy]-phenol (NP65), on Sertoli cells in vitro. Sertoli TM4 cells were exposed to various concentration (0, 0.1, 1, 10, or 20μM) of NP65 for 24h, and the outcomes indicated that treatment of NP65 induced reactive oxygen species (ROS) generation, oxidative stress, and apoptosis for Sertoli TM4 cells. In addition, it was found that NP65 exposure affected homeostasis of Ca2+ in Sertoli TM4 cells by increasing cytoplasm [Ca2+]i, inhibiting Ca2+-ATPase activity and decreasingcyclic adenosine monophosphate (cAMP) concentration. Pretreatment with ROS scavenger, N-acetylcysteine (NAC), attenuated NP65-induced oxidative stress as well as apoptosis for TM4 cells. Furthermore, NAC blocked NP65-induced disorders of Ca2+ homeostasis by attenuating the growth of intracellular [Ca2+]i and the inhibition of Ca2+-ATPase and cAMP activities. Thus, we have demonstrated that NP65 induced apoptosis as well as acted as a potent inhibitor of Ca2+-ATPase activity and resulted in disorder of Ca2+ homeostasis in Sertoli TM4 cells; ROS participated in the process. Our results supported the view that oxidative stress acted an essential role within the development of apoptosis and Ca2+ overload in TM4 cells as a consequence of NP65 stimulation.

QuEChERS-Based Methodology for the Screening of Alkylphenols and Bisphenol A in Dairy Products Using LC-LTQ/Orbitrap MS

A simple methodology was developed for the determination of four Endocrine Disrupting Chemicals (EDCs) in dairy products. The EDCs included alkylphenols (4-tert-octylphenol, technical nonylphenol isomers, 4-nonylphenol) and bisphenol-A. The methodology consisted of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction followed by liquid chromatography (LC) coupled to the hybrid LTQ/Orbitrap mass spectrometer (MS). The high resolution (HR) analysis provided the required selectivity demonstrating excellent sensitivity and enabled the high-mass accuracy of the analytes within short time of analysis, after a chemometric optimization of the instrument parameters. An experimental design was employed for the estimation of the effect of different parameters on the QuEChERS extraction efficiency to obtain the optimum conditions. Method validation proved that analysis exhibited excellent linearity (R2 > 0.9966), low enough precision (0.6 to 13.3%) and recoveries in the range of 91 to 108%. Limits of detection (LOD < 6.5 ng g−1) and quantification (LOQ < 20 ng g−1) as well as matrix effects (ME) were also evaluated. The optimized method was successfully applied to analyze dairy commodities varying in fat content and packaging material including milk, yogurts and infant formulae. Detected concentration levels (MDL-10.4 ng g−1) for bisphenol-A BPA in milk samples resulted in 0.36% of TDI for the medium case (average BPA concentrations) and 1.15% of TDI for the worst case (maximum BPA concentration).

Degradation, transformation, and non-extractable residue formation of nitrated nonylphenol isomers in an oxic soil

Nitrated nonylphenols (NNPs) are main metabolites of the endocrine-disrupting nonylphenols in soil, yet their fate is unknown. Here, using four NNP isomers (NNP111, NNP112, NNP65, and NNP38), the degradation pattern of NNPs was investigated in an oxic soil for 266 days. Specifically, NNP111 was 14C-labeled to facilitate investigating its degradation, transformation, and non-extractable residue (NER) formation. NNPs degradation was isomer-specific with the decreasing order of half-life: NNP111 (126 days) > NNP112 (76 days) > NNP65 (14 days) > NNP38 (8.4 days), providing direct evidence of the greater persistence of NNPs in soil than their parent NPs. At the end of the incubation, 8.5 %, 7.3 %, and 39.9 % of 14C-NNP111 was mineralized, transformed to 2-amino-NP111, and formed NERs in active soil, respectively. In contrast, NERs in sterilized soils were significantly lower, amounting to 15.1 % and 17.3 % in autoclaved and γ-irradiated soil, respectively. The majority of the NERs (>70 %) were in humin fraction, in which type I NER was the predominant (>90 %) mode for NER formation. Our results provide comprehensive knowledge on the fate of NNPs in soil, demonstrating that isomer-specific behavior, transformation products of NNPs, and NER formation should be considered when evaluating environmental fate and risks of NNPs.

Influence of nonylphenol from multilayer plastic films on artificial insemination of sows.

Artificial insemination is common practice in mass livestock farming. Recently, it was shown that chemicals leaching from multilayer plastic bags affect the fertility of boars, although common quality tests did not show any impact on the sperm. It is not clear whether this incidence was a single case or whether it could be a systematic problem. Therefore, we studied six multilayer plastic bags. A total of 49 compounds were found, but most of them were at very low intensity. Nonylphenols in the range of 19-99μg/g plastic were found. Migration tests using water and 10% ethanol as simulants, to mimic the behavior of semen with the extender, were performed. The most interesting migrants in terms of potential reprotoxicity were identified as nonylphenols. The identification in depth demonstrated the presence of 10 isomers of nonylphenol with a total concentration range between 16 to 58μg/Kg simulant, among other migrants at lower concentration. The influence of these nonylphenols and their maximum tolerable concentration in direct contact with semen from boars was studied. Motility, viability, mitochondrial activity and acrosomes reacted were significantly affected at 10mg/Kg of nonylphenols in contact with the sperm, but in vitro penetration rate was significantly decreased with only 2mg/Kg. Insight into the mode of action is also provided.

Intergenerational toxicity of nonylphenol ethoxylate (NP-9) in Caenorhabditis elegans.

The ethoxylated isomers of nonylphenol (NPEs, NP-9) are one of the main active ingredients present in nonionic surfactants employed as herbicides, cosmetics, paints, plastics, disinfectants and detergents. These chemicals and their metabolites are commonly found in environmental matrices. The aim of this work was to evaluate the intergenerational toxicity of NP-9 in Caenorhabditis elegans. The lethality, length, width, locomotion and lifespan were investigated in the larval stage L4 of the wild strain N2. Transgenic green fluorescent protein (GFP) strains were employed to estimate changes in relative gene expression. RT-qPCR was utilized to measure mRNA expression for neurotoxicity-related genes (unc-30, unc-25, dop-3, dat-1, mgl-1, and eat-4). Data were obtained from parent worms (P0) and the first generation (F1). Lethality of the nematode was concentration-dependent, with 48 h-LC50 values of 3215 and 1983μM in P0 and F1, respectively. Non-lethal concentrations of NP-9 reduced locomotion. Lifespan was also decreased by the xenobiotic, but the negative effect was greater in P0 than in F1. Non-monotonic concentration-response curves were observed for body length and width in both generations. The gene expression profile in P0 was different from that registered in F1, although the expression of sod-4, hsp-70, gpx-6 and mtl-2 increased with the surfactant concentration in both generations. None of the tested genes followed a classical concentration-neurotoxicity relationship. In P0, dopamine presented an inverted-U curve, while GABA and glutamate displayed a bimodal type. However, in F1, inverted U-shaped curves were revealed for these genes. In summary, NP-9 induced intergenerational responses in C. elegans through mechanisms involving ROS, and alterations of the GABA, glutamate, and dopamine pathways.

Degradation and transformation of nitrated nonylphenol isomers in activated sludge under nitrifying and heterotrophic conditions

Nitrated nonylphenols (2-nitro-nonylphenols, NNPs) are metabolites of the endocrine-disrupter nonylphenols (NPs). While they have been detected in the environment, their fate in activated sludge has yet to be determined. In this study, we used synthesized NNP isomers and a 14C-tracer technique to study the degradation and transformation of four NNP isomers (NNP111, NNP112, NNP38, and NNP65) in nitrifying activated sludge (NAS) and heterotrophic bacteria-enhanced activated sludge (HAS). Our results showed that the degradation of NNPs in both NAS and HAS was isomer-specific. The half-lives of the NNPs decreased in the order: NNP111 > NNP112 > NNP38 > NNP65. After 36 days of incubation, 9.48 % and 4.01 % of the 14C-NNP111 was mineralized in NAS and HAS, respectively. In addition to mineralization, five metabolites of NNPs containing hydroxyl, carbonyl, and carboxyl substituents on the alkyl chains were formed in NAS but not in HAS. The transformation of NNPs differed in NAS and HAS, mainly due to the differences in their microbial communities and the activities thereof in NAS and HAS. This is the first study of the isomer-specific fate of NNP isomers in activated sludge. Future studies should assess the toxicity, stability and potential risks of NNP metabolites in the environment.

Molecular docking simulation on the interactions of laccase from Trametes versicolor with nonylphenol and octylphenol isomers.

The biodegradation of nonylphenol (NP) and octylphenol (OP) isomers by laccase has attracted increasing concerns. However, the interaction mechanism between these isomers and laccase remains unclear, especially for fungal laccase. In this work, molecular docking was employed to study this issue. The results indicated that the structural characteristic of alkyl chain (position and branching degree) affected the interactions between Trametes versicolor (T. versicolor) laccase and isomers. The binding affinity between them was closely related to the position and branching degree of alkyl chain in isomers. The binding affinities between linear isomers and T. versicolor laccase were para-position < meta-position < ortho-position. For selected branched 4-NP, the isomers with bulky α-substituent in alkyl chain had higher binding affinities. In addition, hydrophobic contacts between T. versicolor laccase and NP or OP isomers were necessary, while H-bonds were optional. The isomers with similar structure may have more common residues involved in hydrophobic contacts. The H-bonds of selected NPs and OPs were all connected with phenolic hydroxyl. These findings provide an insight into detailed interaction mechanism between T. versicolor laccase and isomers of NP and OP. It is helpful to broaden the knowledge of degradation technology of NPs and OPs and provide theoretical basis on biological remediation of these contaminants.

Generalized concentration addition accurately predicts estrogenic potentials of mixtures and environmental samples containing partial agonists

Cell-based bioanalytical tools are considered one alternative to overcome limitations of sensitivities of instrumental, analytical chemistry for monitoring estrogenic chemicals in the environment. Because these tools also reflect non-additive interactions of chemicals in mixtures, their outcomes often deviate from outcomes of chemical analytical approaches that assume additivity, e.g. the concentration addition (CA) model. Often this is because CA is unable to adequately represent effects of partial agonists, i.e. estrogens with lesser efficacies compared to 17β-estradiol. A generalized concentration addition (GCA) model has been proposed to address this shortcoming. In the present study, we investigated effects of mixtures of isomers of nonylphenol as partial model agonists in a cell-based estrogenicity assay. Whether the GCA model was able to more accurately predict the outcomes of these and previously published mixture experiments was evaluated, as well as the potency of a set of comprehensively characterized sewage effluent samples, compared to CA. If samples contained partial agonists, the GCA model consistently predicted potencies of mixtures and extracts of environmental samples more accurately than did the CA model. These findings enable more accurate estimations of potencies of estrogenicity explained by concentrations of agonists and partial agonists, thus significantly improving the ability to identify causative chemicals.

Effect of rice-straw biochar on selective biodegradation of nonylphenols in isomer specificity.

In a previous study, we found that rice-straw biochar degraded and removed hydrophobic organic contaminants (HOCs) through coupled adsorption-biodegradation. However, few studies have determined whether biochar affects HOC isomer degradation and isomer-selective biodegradation or whether biochar can alter HOC isomer features, resulting in changes to HOC isomer residues in water environments. In this study, the effects of biochar at two dosages (0.001 and 0.01g) on the biodegradation of ten isomers of a typical xenoestrogen of nonylphenol (NP) were evaluated. The results revealed that there were no effects of biochar on the adsorption of NP isomers. However, biochar addition affected the biodegradation of a specific isomer without altering the features of the NP isomers. The treatment of NP isomers with Pseudoxanthomonas sp. yielded degradation ratios ranging from 60.7 to 100%. At 0.001 g biochar treatment, the degradation of eight NP isomers was enhanced (except for NP194 and NP193a+b) due to their bulky structures. The degradation of the ten NP isomers was inhibited when 0.01 g biochar was added. These findings characterized the effects of biochar on NP isomer contaminants and provided basic information for the application of biochar for the remediation of NP isomer contaminants.

Determining the Residual Characteristics of Alkylphenols, Arsenic, and Lead as well as Assessing the Exposures of 1,4-Dioxane from Household Food Detergents.

Food detergents are commonly used in households. The main components of detergents include surfactants and water. It has been found that certain compounds, which may cause health concerns, appear in food detergents, such as alkylphenols, arsenic, and lead. After applying food detergents is when people may be exposed to various levels of these chemicals when there are residues. In this study, the Taguchi experimental design was performed to determine the possible factors that might affect the residual characteristics of food detergent on dishware, fruits, and vegetables. The results showed that the variety of detergent was found to be the most significant factor affecting the residue amounts of arsenic (62.9%) and lead (71.6%) on fruits and vegetables, whereas the concentration of detergent used affected the amount of lead residue only (10.5%). On the other hand, dishware material, the concentration of analytes, immersion time, and type of surfactant contributed to arsenic residues on dishware, whereas technical nonylphenol isomer residues on dishware increased as the concentration of spiked analyte increased. In addition, the occurrence of 1,4-dioxane, a possible human carcinogen, in household food detergents in Taiwan was also determined in this research by solid-phase microextraction and GC-MS. Among the 80 detergent samples, 71 contained different concentrations ranges of 1,4-dioxane, from 0.03 to ~3.73 µg/g. In the exposure assessment, it was estimated that the maximum amounts of 1,4-dioxane in contact with the skin from the use of household food detergent in Taiwan was 0.015 µg/kg/day.

Alkylphenol and phthalate contamination of all sources of greywater from French households

Despite the importance of domestic wastewater, the contribution of greywater to both alkylphenol and phthalate pollution is not yet well documented. Likewise, the detailed emissions of phthalates and alkylphenols by greywater have been insufficiently studied, this work aims to fill this gap. The levels of four phthalates (DEP, DnBP, BBP and DEHP) and two alkylphenols (nonylphenol isomers and octylphenol) were quantified in six different types of greywater, namely that from washing machines, manual dishwashing, dishwashers, bathroom water (from showers and sinks) and floor cleaning. This paper presents the methodology used to characterize all sources of greywater and provides their levels of contamination. The highest concentrations were found in greywater produced by the washing machine and floor cleaning, while washing machine and shower greywater have the highest phthalate and alkylphenol loads because of the volume associated with these two sources of greywater.

An Isomer-Specific Approach to Endocrine-Disrupting Nonylphenol in Infant Food

Nonylphenols (NPs) are persistent endocrine disruptors that are priority hazardous substances of the European Union Water Framework Directive. Their presence in the environment has caused growing concern regarding their impact on human health. Recent studies have shown that nonylphenol is ubiquitous in commercially available foodstuffs and is also present in human blood. The isomer distribution of 4-nonylphenol was analyzed by gas chromatography - mass spectrometry in 44 samples of infant food. Our study shows that the distribution of nonylphenol isomers is dependent on the foodstuff analyzed. Although some isomer groups prevail, different distributions are frequent. Variations are even found in the same food group. Nonylphenol is a complex mixture of isomers, and the estrogenic potentials of each of these isomers are very different. Consequently, to determine the potential toxicological impact of NP in food, an isomer-specific approach is necessary.

Enantioselective separation of defined endocrine-disrupting nonylphenol isomers.

Nonylphenol is in the focus of worldwide endocrine-disrupter research and accountedfor as a priority hazardous substance of the Water Framework Directive of the European Union. Technical nonylphenol consists of a very complex mixture of isomers and enantiomers. As estrogenic effect and degradation behavior in environmental processes of single nonylphenols are heavily dependent on the structure of the nonyl side chain, it is absolutely necessary to consider the nonylphenol problem from an isomer and enantiomer-specific viewpoint. In this study, an enantiomer-specific separation of eight defined synthesized nonylphenol isomers by five different special chiral cyclodextrin columns was performed underivatized and after methylation, silylation, and acylation. This work demonstrates that three columns out of the investigated five show an excellent separation behavior for the studied different nonylphenol isomers and can be used for the enantiomer-specific determination of nonylphenols in food, other biological matrices, and environmental samples in the future. Graphical abstract Enantiomeric pair of 4-NP170 (4-[1-ethyl-1,3,3-trimethylbutyl]phenol).

Downregulation of steroid hormone receptor expression and activation of cell signal transduction pathways induced by a chiral nonylphenol isomer in mouse sertoli TM4 cells.

Nonylphenols (NPs) are considered as important environmental toxicants and potential endocrine disrupting compounds which can disrupt male reproductive system. 4-[1-Ethyl-1-methylhexy] phenol (4-NP65 ) is one of the main isomers of technical nonylphenol mixtures. In the present study, effect of NPs was evaluated from an isomer-specific viewpoint using 4-NP65 . Decreased mRNA expression levels of estrogen receptor (ER)-α, ER-β, androgen receptor (AR) and progesterone receptor (PR) were observed in the cells exposed to 4-NP65 for 24 h. Furthermore, 4-NP65 treatment evoked significant decrease in protein expression levels of ER-α and ER-β. Levels of mullerian inhibiting substance and transferrin were found to change significantly in 4-NP65 challenged cells. Additionally, JNK1/2-MAPK pathway was activated due to 4-NP65 exposure, but not ERK1/2 and p38-MAPK pathways. Meanwhile, 4-NP65 increased the p-Akt level and showed no effects on the Akt level which indicated that Akt pathway was activated by 4-NP65 . In conclusion, these findings have shown that 4-NP65 exposure affected expression of cell receptors and cell signaling pathways in Sertoli TM4 cells. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signal transduction pathways, and that the effects were dependent on the side chain of NP isomers. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 469-476, 2017.