Aniline Exposure Research Articles

Evaluation of haematological, genotoxic, cytotoxic and ATR-FTIR alterations in blood cells of fish Channa punctatus after acute exposure of aniline

Aniline (C6H5NH2) an important intermediate in the organic and fine chemical industry, is ubiquitously used worldwide. It is one of the important building block for manufacturing of 4,4-methylene diphenyl diisocyanate (MDI), accelerators in rubber processing, dyes, tattoo inks, photographic chemicals, antioxidants, corrosion inhibitors, pharmaceuticals and antiseptics. The current study evaluated 96 h LC50 of aniline and based on this, two sublethal concentrations (4.19 mg/l and 8.39 mg/l) were selected for acute exposure studies in freshwater food fish Channa punctatus. Erythrocytes of fish are nucleated hence they play an important role in physiology, immune system, protein signalling and haemostatic condition along with respiration. Blood samples were collected after 24, 48, 72, and 96 h of exposure to study haematological, cytotoxic and genotoxic effects of sublethal concentrations of aniline in C. punctatus. Symbolic elevation in time and dose dependent DNA damage was observed by comet assay as well as micronuclei assay revealing maximum damage after 96 h of exposure. After aniline exposure, scanning electron microscopy and ATR-FTIR studies showed anomalies in structure and alterations in biomolecules of RBCs of aniline exposed group as compared to control group respectively. Semi prep HPLC studies revealed bioaccumulation potential of aniline in higher concentration exposed group.

Aniline exposure activates receptor-interacting serine/threonineprotein kinase 1 and causes necroptosis of AML12 cells.

With the increased use of aniline, potential impacts on human health cannot be ignored. The hepatotoxicity of aniline is largely unknown and the underlying mechanism remains unclear. Therefore, the aim of the present study was to investigate the hepatotoxicity of aniline and elucidate the underlying mechanism. AML12 cells were exposed to different concentrations of aniline (0, 5, 10, or 20mM) to observe changes to reactive oxygen species (ROS) production and the expression patterns of necroptosis-related proteins (RIPK1, RIPK3, and MLKL). The potential mechanism underlying aniline-induced hepatotoxicity was explored by knockout of RIPK1. The results showed that aniline induced cytotoxicity in AML12 cells in a dose-dependent manner in addition to the production of ROS and subsequent necroptosis of AML12 cells. Silencing of RIPK1 reversed upregulation of necroptosis-related proteins in AML12 cells exposed to aniline, demonstrating that aniline-induced ROS production was related to necroptosis of AML12. Moreover, aniline promoted intracellular RIPK1 activation, suggesting that the RIPK1/ROS pathway plays an important role in aniline-induced hepatotoxicity. NAC could quench ROS and inhibit necroptosis. These results provide a scientific basis for future studies of aniline-induced hepatotoxicity for the prevention and treatment of aniline-induced cytotoxicity.

A bi-functionalized metal-organic framework based on N-methylation and Eu3+ post-synthetic modification for highly sensitive detection of 4-Aminophenol (4-AP), a biomarker for aniline in urine

4-Aminophenol (4-AP), which is a biomarker of aniline and represents the internal dose of aniline exposure in the human body, has attracted much attention for its detection in recent years. In this work, a bi-functionalized luminescent metal-organic framework (MOF), Eu@MOF-253-CH3, is designed and prepared through encapsulating the methyl groups and the Eu3+ cations into MOF-253 based on post-synthetic modification strategy. This study shows that the bi-functionalized Eu@MOF-253-CH3 can specifically recognize 4-AP upon luminescence quenching, while refraining from the interference of other coexisting species in urine. The Eu@MOF-253-CH3 hybrid as a 4-AP sensor also displays excellent performances including high water tolerance, good pH-independent stability, fast response, great selectivity and elevated sensitivity (0.5 μg mL−1) attributed to N-viologenized ligand. These results suggest the bi-functionalized Eu@MOF-253-CH3 can act as a promising sensor to practically monitor 4-AP's concentrations in human urine system, and then to realize the screening and pre-diagnosis of human health. Moreover, the possible sensing mechanisms are further explored at length.

A novel inhibition mechanism of aniline on nitrification: Aniline degradation competes dissolved oxygen with nitrification

Aniline is a toxic aromatic amine and an inhibitor of nitrification. This study explored the inhibition effect and underlying mechanism. After sludge acclimation, 540 mg/L aniline was removed in 24 h and almost all ammonia released from aniline was oxidized to nitrate. However, nitrification never started until no aniline left. The cellular adenosine triphosphate (cATP) concentration of acclimated sludge reduced only by 2% after aniline exposure. Neither transmembrane transport of ammonia nor ammonia monooxygenase (AMO) activity was affected by aniline. Growing initial aniline concentration did not deteriorate the specific nitrification rate (NR). These all revealed that the toxicity of aniline only play a minor role in inhibition. Competition for dissolved oxygen (DO) was proposed to be another possible inhibition mechanism. The oxygen affinity constant (Ks) of aniline degraders and ammonia-oxidizing bacteria (AOB) was calculated to be 0.894 mg/L and 1.274 mg/L respectively, suggesting the former possessed much stronger oxygen affinity (P < 0.01). With aniline and ammonium as initial substrates, increasing aeration intensity advanced nitrification and increased the NR. Max NR of 0.63 mgN/(gMLSS·h) was achieved at the highest aeration intensity of 1000 mL/min. This study brings one step closer to better removal of aniline and derived nitrogen pollutants.

Salinity relief aniline induced oxidative stress in Suaeda salsa: Activities of antioxidative enzyme and EPR measurements

Wastewater from printing and dyeing processes often contains aniline and high salinity, which are hazardous to aquatic species. Glycophytic plants cannot survive under high-salinity conditions, whereas halophytes grow well in such an environment. In this study, we investigated the influence of NaCl on the antioxidant level in Suaeda salsa affected by aniline stress. The seedlings showed various growth toxicity effects under different concentrations of aniline. The results showed that the effect of the aniline was more severe for the root growth compared to that for the shoot growth. Aniline exposure significantly increased the total free radicals and ·OH radicals in the plants. Suaeda salsa exposure to aniline caused oxidative stress by altering the superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity, which resulted in the overproduction of H2O2 and the inducement of lipid peroxidation. Analysis revealed that the malondialdehyde (MDA) content was enhanced after aniline exposure and that the chlorophyll content was significantly decreased. The results showed that aniline induced the production of free radicals and reactive oxygen species (ROS), and changed the antioxidant defense system. This ultimately resulted in oxidative damage in S. salsa; however, it was found that moderate salinity could mitigate the effects. In conclusion, salinity may alleviate the growth inhibition caused by aniline by regulating the antioxidant capacity of S. salsa.

Research progress on health effects of aniline

Aniline is one of the important chemical raw materials in daily life and the chemical industry. Aniline exposure might occur through intact skin, respiratory tract and digestive tract. It could pose negative impacts on many organs and systems of the human body, including toxicity or carcinogenicity to blood, liver, and spleen. This paper summarized the direct effects of aniline on human health and the indirect hazards of aniline on human health through environmental pollution and discussed the future research directions of aniline-induced health hazards.

New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2

Aniline is a major environmental pollutant of serious concern due to its toxicity. Although microbial metabolism of aniline is well-studied, its toxic effects and physiological responses of microorganisms to aniline are largely unexplored. Rubrivivax benzoatilyticus JA2, an aniline non-degrading bacterium, tolerates high concentrations of aniline and produces extracellular polymeric substance(EPS). Surprisingly, strain JA2 forms EPS only when exposed to aniline and other toxic compounds like organic solvents and heavy metals indicating that EPS formation is coupled to cell toxicity. Further, extensive reanalysis of the previous proteomic data of aniline exposed cells revealed up-regulation of envelope stress response(ESR) proteins such as periplasmic protein folding, envelope integrity, transmembrane complex, and cell-wall remodelling proteins. In silico analysis and molecular modeling of three highly up-regulated proteins revealed that these proteins were homologous to CpxARP proteins of ESR signalling pathway. Furthermore, EPS formation to known ESR activators(Triton-X-100, EDTA) suggests that envelope stress possibly regulating the EPS production. The present study suggests that aniline triggers envelope stress; to counter this strain JA2 activates ESR pathway and EPS production. Our study revealed the hitherto unknown toxic effects of aniline as an acute envelope stressor thus toxicity of aniline may be more profound to life-forms than previously thought.

Molecular Mechanism of Aniline Induced Spleen Toxicity and Neuron Toxicity in Experimental Rat Exposure: A Review.

Aniline exposure leads to neuron and spleen toxicity specifically and makes diverse neurological effects and sar-coma that is defined by splenomegaly, hyperplasia, and fibrosis and tumors formation at the end. However, the molecular mechanism(s) of aniline-induced spleen toxicity is not understood well, previous studies have represented that aniline expo-sure results in iron overload and initiation of oxidative/nitrosative disorder stress and oxidative damage to proteins, lipids and DNA subsequently, in the spleen. Elevated expression of cyclins, cyclin-dependent kinases (CDKs) and phosphorylation of pRB protein along with increases in A, B and CDK1 as a cell cycle regulatory proteins cyclins, and reduce in CDK inhibitors (p21 and p27) could be critical in cell cycle regulation, which contributes to tumorigenic response after aniline exposure. Aniline-induced splenic toxicity is corre-lated to oxidative DNA damage and initiation of DNA glycosylases expression (OGG1, NEIL1/2, NTH1, APE1 and PNK) for removal of oxidative DNA lesions in rat. Oxidative stress causes transcriptional up-regulation of fibrogenic/inflammatory factors (cytokines, IL-1, IL-6 and TNF-α) via induction of nuclear factor-kappa B, AP-1 and redox-sensitive transcription factors, in aniline treated-rats. The upstream signalling events as phosphorylation of IκB kinases (IKKα and IKKβ) and mito-gen-activated protein kinases (MAPKs) could potentially be the causes of activation of NF-κB and AP-1. All of these events could initiate a fibrogenic and/or tumorigenic response in the spleen. The spleen toxicity of aniline is studied more and the different mechanisms are suggested. This review summarizes those events following aniline exposure that induce spleen tox-icity and neurotoxicity.

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

Human metabolism and excretion kinetics of aniline after a single oral dose.

Aniline is an important source material in the chemical industry (e.g., rubber, pesticides, and pharmaceuticals). The general population is known to be ubiquitously exposed to aniline. Thus, assessment of aniline exposure is of both occupational and environmental relevance. Knowledge on human metabolism of aniline is scarce. We orally dosed four healthy male volunteers (two fast and two slow acetylators) with 5mg isotope-labeled aniline, consecutively collected all urine samples over a period of 2days, and investigated the renal excretion of aniline and its metabolites by LS-MS/MS and GC-MS. After enzymatic hydrolysis of glucuronide and sulfate conjugates, N-acetyl-4-aminophenol was the predominant urinary aniline metabolite representing 55.7-68.9% of the oral dose, followed by the mercapturic acid conjugate of N-acetyl-4-aminophenol accounting for 2.5-6.1%. Acetanilide and free aniline were found only in minor amounts accounting for 0.14-0.36% of the dose. Overall, these four biomarkers excreted in urine over 48h post-dose represented 62.4-72.1% of the oral aniline dose. Elimination half-times were 3.4-4.3h for N-acetyl-4-aminophenol, 4.1-5.5h for the mercapturic acid conjugate, and 1.3-1.6 and 0.6-1.2h for acetanilide and free aniline, respectively. Urinary maximum concentrations of N-acetyl-4-aminophenol were reached after about 4h and maximum concentrations of the mercapturic acid conjugate after about 6h, whereas concentrations of acetanilide and free aniline peaked after about 1h. The present study is one of the first to provide reliable urinary excretion factors for aniline and its metabolites in humans after oral dosage, including data on the predominant urinary metabolite N-acetyl-4-aminophenol, also known as an analgesic under the name paracetamol/acetaminophen.

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen.

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a tumorigenic response on chronic aniline exposure.

Oxidative Stress Induced Following Exposure to 3,5‐Dichloroaniline (3,5‐DCA) In Vitro: Role in Nephrotoxicity

Anilines and their chlorinated derivatives are used in the production of a wide variety of agricultural products, drugs and dyes. Studies have shown that chlorinated aniline exposure leads to nephrotoxicity in both in vitro and in vivo models. Of the mono‐ and dichloroanilines studied, 3,5‐dichloroaniline (3,5‐DCA) proved to posses the most nephrotoxic potential. Exposure to 1.0 mM 3,5‐DCA for 90 min significantly increased LDH release in isolated renal cortical cells (IRCC) from male Fischer 344 rats. Pretreatment of IRCC with antioxidants significantly attenuated LDH release, suggesting that free radical/oxidative stress mechanisms may play a role in 3,5‐DCA induced nephrotoxicity. The purpose of the current study was to explore the potential role of oxidative stress in 3,5‐DCA nephrotoxicity in vitro. IRCC (4 x 106 cells/ml; 3 ml) were exposed to 3,5‐DCA (0, 0.5, or 1.0 mM) for either 60 or 90 min. Aliquots were then taken for LDH release assays, protein carbonyl detection using Oxyblot kits from Millipore, and glutathione (GSH and GSSG) determination assays. Protein carbonyls were significantly increased following exposure to 1.0 mM 3,5‐DCA after 90 min only, while a shift in the ratio of GSSG/GSH was seen at exposure to 1.0 mM 3,5‐DCA for 60 min. However, in both cases the increase in oxidative stress markers occurred after there was a significant increase in LDH release. These findings suggest that despite 3,5‐DCA's ability to induce oxidative stress, oxidative stress is not an early initiator of 3,5‐DCA nephrotoxicity in IRCC. These studies were supported, in part, by NIH grant P20GM103434.

Aniline exposure associated with up-regulated transcriptional responses of three glutathione S-transferase Delta genes in Drosophila melanogaster

Complex transcriptional profile of glutathione S-transferase Delta cluster genes occurred in the developmental process of the fruit fly Drosophila melanogaster. The purpose of this project was to quantify the expression levels of Gst Delta class genes altered by aniline exposure and to understand the relationship between aniline dosages and the variation of Gst Delta genes expressed in D. melanogaster. Using RT-PCR expression assays, the expression patterns of the transcript mRNAs of the glutathione S-transferase Delta genes were revealed and their expression levels were measured at eggs, larvae, pupae and adults. The adult stage was selected for further dose–response assays. After analysis, the results indicated that three Gst Delta genes (Gst D2, Gst D5 and Gst D6) were found to show a peak of up-regulated transcriptional response at 6–8h of exposure of aniline. Furthermore, the dose–response relationship of their induction levels within the dose regiments (from 1.2 to 2.0μl/tube) had been measured. The expression patterns and annotations of these genes were discussed in the context.

Ubiquitous presence of paracetamol in human urine: sources and implications

N-acetyl-4-aminophenol (acetaminophen/paracetamol, NA4AP) is one of the most commonly used over-the-counter analgesic and antipyretic drugs. Recent studies have reported anti-androgenic effects of NA4AP in vitro and possible associations between intrauterine exposure to NA4AP and the development of male reproductive disorders in humans. NA4AP is also a major metabolite of aniline (phenylamine), representing 75-86% of the aniline dose excreted in urine. Aniline is an important large-volume intermediate in several industrial processes. Besides individuals in various occupational settings with aniline exposure, the general population is also known to be ubiquitously exposed to aniline. In this article, we provide an overview of the recent literature concerning the intake of NA4AP during pregnancy and the possible anti-androgenic effects of NA4AP as well as literature concerning its known metabolic precursor aniline. We also present new research data, including the first human biomonitoring data on NA4AP excretion in urine, showing ubiquitous NA4AP body burdens in the general population at a wide range of concentrations. We found a small but significant impact of smoking on urinary NA4AP concentrations. We further present preliminary data on NA4AP excretion after therapeutic acetaminophen use, after aniline exposure in an occupational setting, and during a controlled fasting study (excluding oral exposure to both aniline and acetaminophen). Our findings indicate exposure to aniline (or aniline-releasing substances) as well as nutrition (next to the direct use of acetaminophen as medication) as possible sources of internal body burdens of NA4AP.

Human-Biomonitoring of N-acetyl-4-aminophenol (paracetamol/acetaminophen) - the major metabolite of aniline

Aniline is an important building-block in several industrial processes (e.g. rubber, colorants, pesticides and pharmaceuticals). Exposure to aniline is therefore both of occupational and environmental relevance and the general population is known to be ubiquitously exposed to aniline. N-acetyl-4-aminophenol (NAAP) is the major urinary metabolite of aniline representing 56%-76% of the dose. Furthermore, NAAP, also known under the trade names acetaminophen/paracetamol, is one of the most commonly used over-the-counter analgesics. Recent epidemiological studies suggest that intrauterine exposure to NAAP is a risk factor for the development of male reproductive disorders. Human biomonitoring data on the body burden of the general population (and potentially exposed subpopulations) to NAAP is literally nonexistent. To determine the internal NAAP exposure of the general population, workers exposed to aniline and users of acetaminophen, we developed a fast on-line HPLC-MS/MS- method with isotope dilution quantification of NAAP in urine with a limit of quantification (LOQ) of 0.75 µg/L. In a pilot study, urine samples of 21 volunteers, not occupationally exposed to aniline, were analyzed for NAAP. NAAP was detected in all samples in a wide concentration range between 8.7 µg/L and 22100 µg/L (median 85.7 µg/L). The highest concentration was measured in a volunteer who took acetaminophen approximately a day ago. Half of the volunteers quoted to either never have taken acetaminophen or not during the several weeks before sampling. Therefore, other routes of exposure than direct use of acetaminophen, like aniline exposure or acetaminophen contaminated foodstuff, leading to the NAAP excretions have to be taken into account.

Molecular Mechanism of Aniline induced bladder cancer

The National Institute for Occupational Safety and Health based in Cincinnati, Ohio, reported that an incidence of the bladder cancer risk was 6.5 times higher for workers considered to have definite exposure to ortho‐toluidine and aniline, and 4 times higher for workers with possible exposure among workers employed at a rubber chemical manufacturing plants. Aniline exposure has been shown to cause: i) An increase in iron overload and induction of oxidative stress in the spleen; ii) Enhanced transcriptional and translational up regulation of IL‐1, IL‐6, and TNF‐α. Increased activation of redox‐sensitive transcription factors NF‐κβ and AP‐1. iii) MAPK phosphorylation, AP‐1 activation, and enhanced TGF‐α gene expression. iv) increased expression of heme oxygenase‐1 (HO‐1), contributing pro‐oxidant mechanism, mediated through iron release, accumulation thus leading to oxidative damage. v) Increased expression of cyclins, Cyclin dependant Kinases (CDKs) and phosphorylation of retinoblastoma protein (pRB) protein; vi) Increases in both p‐IKKα and p‐IKK£]. Taken together, it is suggested that above sequence of events could represent the plausible underlying mechanism of aniline exposure induced oxidative stress.

Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure

Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5mmol/kg/day via drinking water for 30days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.

Aniline-induced nitrosative stress in rat spleen: Proteomic identification of nitrated proteins

Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6 kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity.

Induction of NEIL1 and NEIL2 DNA glycosylases in aniline-induced splenic toxicity

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2′-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7- and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0- and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline-induced oxidative stress is associated with an induction of NEIL1/2. The increased NIEL-mediated BER activity is another indication of aniline-induced oxidative damage in the spleen and could constitute another important mechanism of removal of oxidative DNA lesions, especially in transcribed DNA following aniline insult.

Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen

Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in the spleen.