Synthesis and evaluation of styrene-maleic acid copolymer conjugated amphotericin B.

Amphotericin B (AmB), which plays a central role in the treatment of systemic fungal infections, is difficult to formulate because it's sparingly soluble in water and organic solvents. We previously prepared AmB-loaded micelles using styrene-maleic acid copolymer (SMA). Although solubilization was achieved by this formulation, stability in the blood circulation was as insufficient as that of Fungizone®, which is a conventional formulation of AmB. Meanwhile, it is well known that polymer-drug conjugates are more stable in circulation than drug-loaded micelles. Therefore, in this study, we developed covalently conjugated SMA-AmB (SMA-AmB conjugate). The SMA-AmB conjugate was found to be soluble and present as micelles in aqueous solution. Furthermore, it was revealed that this micelle behaves as a larger molecule by forming a complex with albumin. The circulation in the blood increased significantly compared to that of Fungizone®, which was suggested to be due to this complex-forming ability. Although in vitro and in vivo antifungal activity of the SMA-AmB conjugate against Saccharomyces cerevisiae was reduced by 1/3 compared to that of Fungizone®, hemolysis decreased to 1/40 or less, and the LD50 decreased to 1/10. In conclusion, it is expected that the SMA-AmB conjugate can be a polymer-therapeutic agent with high antifungal selectivity.

Human plasma and liver concentrations of styrene estimated by combining a simple physiologically based pharmacokinetic model with rodent data.

Long-term exposure to certain volatile organic compounds is a significant public health concern. A variety of food containers and drinking cups prepared from polystyrene or polystyrene-related plastics could contain styrene monomer. In the current study, the concentrations of styrene in plasma and liver were surveyed and determined after oral doses of 25 mg/kg to rats and 200 mg/kg to control and humanized-liver mice. Plasma concentrations of styrene in rats were still detected 2 hr after 10-25 mg/kg oral doses. In contrast, after an order of magnitude higher oral dose of styrene (200 mg/kg) to mice, styrene in mouse plasma was rapidly cleared within 15 min to the limit-of-detection level. However, unmetabolized styrene was detected in mouse liver 24 hr after oral treatment. A simple physiologically based pharmacokinetic (PBPK) model capable of estimating blood and liver concentrations of styrene was established for rats. A human PBPK model was then set up for styrene by using the same intrinsic hepatic clearances in rats and humans and by applying allometric scaling to rat parameters obtained from the plasma concentrations of styrene in rats. By reverse dosimetry analysis (from concentrations to doses), we found that the 95th percentile values of styrene concentrations (0.132 ng/mL) reported in United States biomonitoring data of more than 1000 human blood samples may imply exposure to repeated oral doses of styrene of 2.89 µg/kg/day. These results suggest that styrene biomonitoring data in human blood samples imply exposures roughly similar to or lower than the established tolerable daily intake level of 7.7 µg/kg/day.

Strain-related differences in mouse lung gene expression over a two-year period of inhalation exposure to styrene: Relevance to human risk assessment.

Both CD-1 and C57BL/6 wildtype (C57BL/6-WT) mice show equivalent short-term lung toxicity from exposures to styrene, while long-term tumor responses are greater in CD-1 mice. We analyzed lung gene expression from styrene exposures lasting from 1-day to 2-years in male mice from these two strains, including a Cyp2f2(-/-) knockout (C57BL/6-KO) and a Cyp2F1/2A13/2B6 transgenic mouse (C57BL/6-TG). With short term exposures (1-day to 1-week), CD-1 and C57BL/6-WT mice had thousands of differentially expressed genes (DEGs), consistent with changes in pathways for cell proliferation, cellular lipid metabolism, DNA-replication and inflammation. C57BL/6-WT mice responded within a single day; CD-1 mice required several days of exposure. The numbers of exposure related DEGs were greatly reduced at longer times (4-weeks to 2-years) with enrichment only for biological oxidations in C57BL/6-WT and metabolism of lipids and lipoproteins in CD-1. Gene expression results indicate a non-genotoxic, mouse specific mode of action for short-term styrene responses related to activation of nuclear receptor signaling and cell proliferation. Greater tumor susceptibility in CD-1 mice correlated with the presence of the Pas1 loci, differential Cytochrome P450 gene expression, down-regulation of Nr4a, and greater inflammatory pathway activation. Very few exposure-related responses occurred at any time in C57BL/6-KO or -TG mice indicating that neither the short term nor long term responses of styrene in mice are relevant endpoints for assessing human risks.

Editor's Highlight: Complete Attenuation of Mouse Lung Cell Proliferation and Tumorigenicity in CYP2F2 Knockout and CYP2F1 Humanized Mice Exposed to Inhaled Styrene for up to 2 Years Supports a Lack of Human Relevance.

Styrene is a mouse-specific lung carcinogen, and short-term mode of action studies have demonstrated that cytotoxicity and/or cell proliferation, and genomic changes are dependent on CYP2F2 metabolism. The current study examined histopathology, cell proliferation, and genomic changes in CD-1, C57BL/6 (WT), CYP2F2(-/-) (KO), and CYP2F2(-/-) (CYP2F1, 2B6, 2A13-transgene) (TG; humanized) mice following exposure for up to 104 weeks to 0- or 120-ppm styrene vapor. Five mice per treatment group were sacrificed at 1, 26, 52, and 78 weeks. Additional 50 mice per treatment group were followed until death or 104 weeks of exposure. Cytotoxicity was present in the terminal bronchioles of some CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Hyperplasia in the terminal bronchioles was present in CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Increased cell proliferation, measured by KI-67 staining, occurred in CD-1 and WT mice exposed to styrene for 1 week, but not after 26, 52, or 78 weeks, nor in KO or TG mice. Styrene increased the incidence of bronchioloalveolar adenomas and carcinomas in CD-1 mice. No increase in lung tumors was found in WT despite clear evidence of lung toxicity, or, KO or TG mice. The absence of preneoplastic lesions and tumorigenicity in KO and TG mice indicates that mouse-specific CYP2F2 metabolism is responsible for both the short-term and chronic toxicity and tumorigenicity of styrene, and activation of styrene by CYP2F2 is a rodent MOA that is neither quantitatively or qualitatively relevant to humans.

Evaluation of the mode of action of mouse lung tumors induced by 4-methylimidazole.

4-Methylimidazole (4-MEI) occurs in certain foods and beverages as a product of browning reactions. An increased incidence of lung tumors was reported in mice, but not rats, exposed to levels of 4-MEI in their diet that far exceed human dietary intake. This investigation evaluated the hypothesis that 4-MEI induces mouse lung tumors by the same mode of action (MOA) as styrene: CYP2F2 metabolic activation and increased BrdU labeling. Using styrene (200 mg/kg/day by gavage) as a positive control, histopathology and DNA synthesis (measured by BrdU incorporation) in the bronchiolar region were evaluated in: (1) a 5-day comparative toxicity study in C57BL/6 "wild type" and CYP2F2 "knock out" (KO) mice given 4-MEI at the same dietary concentrations used in the NTP cancer bioassay, and (2) a 13-week comparative toxicity study of C57BL/6 and B6C3F1 mice receiving 0, 1250 or 2500 ppm of 4-MEI in the diet for 6, 15, 34 and 91 days. In contrast to styrene, 4-MEI had no consistent effect on BrdU labeling or histopathology in the lungs of mice in the dose range that had been shown to produce lung tumors in another study. The results of these studies do not support the hypothesis that 4-MEI and styrene induce lung tumors by the same MOA.

Styrene-maleic acid copolymer-encapsulated CORM2, a water-soluble carbon monoxide (CO) donor with a constant CO-releasing property, exhibits therapeutic potential for inflammatory bowel disease.

Carbon monoxide (CO), the physiological product of heme oxygenase during catabolic breakdown of heme, has versatile functions and fulfills major anti-oxidative and anti-apoptotic roles in cell systems. Administration of CO is thus thought to be a reasonable therapeutic approach in diseases-such as inflammatory bowel disease-that are induced by reactive oxygen species (ROS). Tricarbonyldichlororuthenium(II) dimer (CORM2) is a commonly used CO donor, but it has poor aqueous solubility and a very short CO-releasing half-life (t1/2). In the present study, we prepared micelles consisting of water-soluble styrene-maleic acid copolymer (SMA) encapsulating CORM2 (SMA/CORM2) that had a hydrodynamic size of 165.3nm. Compared with free CORM2, SMA/CORM2 demonstrated better water solubility (>50mg/ml in a physiological water solution). Moreover, because of micelle formation in an aqueous environment, the CO release rate was slow and sustained. These properties resulted in much longer in vivo bioactivity of SMA/CORM2 compared with that of free CORM2, i.e. the t1/2 in blood of SMA/CORM2 in mice after intravenous (i.v.) injection was about 35 times longer than that of free CORM2. We then evaluated the therapeutic potential of SMA/CORM2 in a murine model of inflammatory colitis induced by dextran sulfate sodium (DSS). Administration (either i.v. or oral) of SMA/CORM2 once at the beginning of colitis, 3days after DSS treatment, significantly improved colitis symptoms-loss of body weight, diarrhea, and hematochezia-as well as histopathological colonic changes-shortening of the colon and necrosis or ulcers in the colonic mucosa. Up-regulation of inflammatory cytokines including monocyte chemotactic protein-1, tumor necrosis factor-α, and interleukin-6 in this DSS-induced colitis was significantly suppressed in SMA/CORM2-treated mice. SMA/CORM2 may thus be a superior CO donor and may be a candidate drug, which involves cytokine suppression, for ROS-related diseases including inflammatory bowel disease.

Derivation of safe health-based exposure limits for potential consumer exposure to styrene migrating into food from food containers.

Residual styrene present in polystyrene food packaging may migrate into food at low levels. To assure safe use, safe exposure levels are derived for consumers potentially exposed via food using No/Low Adverse Effect Levels from animal and human studies and assessment factors proposed by European organisations (EFSA, ECHA, ECETOC). Ototoxicity and developmental toxicity in rats and human ototoxicity and effects on colour discrimination have been identified as the most relevant toxicological properties for styrene health assessments. Safe exposure levels derived from animal studies with assessment factors of EFSA and ECHA were expectedly much lower than those using the ECETOC approach. Comparable safe exposure levels were obtained from human data with all sets of assessment factors while ototoxicity in rats led to major differences. The safe exposure levels finally selected based on criteria of science and health protection converged to the range of 90-120 mg/person/d. Assuming a consumption of 1 kg food/d for an adult, this translates to 90 mg styrene migration into 1 kg food as safe for consumers. This assessment supports a health based Specific Migration Limit of 90 ppm, a value somewhat higher than the current overall migration limit of 60 ppm in the European Union.

A thermoplastic elastomer patch matrix for traditional Chinese medicine: design and evaluation.

OBJECTIVE: To design and evaluate a novel pressure sensitive adhesive (PSA) patch containing traditional Chinese medicine (TCM) using styrene-isoprene-styrene (SIS) copolymer. METHOD: A mixture D-optimal design with ternary response surface diagram was employed in the optimization process. The proportions of SIS copolymer, tackifying resin and plasticizer were selected as the independent variables while tack force, peel strength of the patch and skin penetrability of methyl salicylate were selected as the dependent variables. The optimized patch was then evaluated including in vivo absorption, pharmacological activities and skin irritation, by comparing with a commercial patch based on natural rubber. RESULTS: The optimized patch, which comprised 30.0% SIS copolymer, 26.6% tackifying resin and 43.4% plasticizer, was superior to commercial patch in skin permeation, pharmacological activities and skin biocompatibility. CONCLUSION: SIS copolymer was a suitable substitute to natural rubber in producing patches containing TCM formula.

Styrene altered clock gene expression in serum-shocked cultured human fibroblasts.

The circadian clock can regulate the metabolic process of xenobiotics, but little is known as to circadian rhythms can be perturbed by xenobiotics. Styrene is a organic chemical widely used in occupational settings. The effects of styrene on the circadian genes of HuDE cells were evaluated after serum-shocking synchronization. A subtoxic dose of 100 µM of styrene altered the expression of clock genes BMAL1, PER2, PER3, CRY1, CRY2, and REV-ERB-α.

DNA damage and susceptibility assessment in industrial workers exposed to styrene.

Styrene is a widely used chemical in the manufacture of synthetic rubber, resins, polyesters, and plastics. The highest levels of human exposure to styrene occur during the production of reinforced plastic products. The objective of this study was to examine occupational exposure to styrene in a multistage approach, in order to integrate the following endpoints: styrene in workplace air, mandelic and phenylglyoxylic acids (MA + PGA) in urine, sister chromatid exchanges (SCE), micronuclei (MN), DNA damage (comet assay), and genetic polymorphisms of metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1, and GSTP1). Seventy-five workers from a fiberglass-reinforced plastics factory and 77 unexposed controls took part in the study. The mean air concentration of styrene in the breathing zone of workers (30.4 ppm) and the mean concentration of urinary metabolites (MA + PGA = 443 ± 44 mg/g creatinine) exceeded the threshold limit value (TLV) and the biological exposure index (BEI). Significantly higher SCE frequency rate and DNA damage were observed in exposed workers, but MN frequency was not markedly modified by exposure. With respect to the effect of genetic polymorphisms on different exposure and effect biomarkers studied, an increase in SCE levels with elevated microsomal epoxide hydrolase activity was noted in exposed workers, suggesting a possible exposure-genotype interaction.

Simultaneous determination of aromatic acid metabolites of styrene and styrene-oxide in rat urine by gas chromatography-flame ionization detection.

A convenient and reliable gas chromatographic method was developed for the simultaneous determination of six aromatic acid metabolites of styrene and styrene-oxide in rat urine; i.e., benzoic (BA), phenylacetic (PAA), mandelic (MA), phenylglyoxylic (PGA), hippuric (HA) and phenylaceturic (PAUA) acids. The method involves a one-pot esterification-extraction procedure, performed directly on urine without prior treatment. Analyses were performed on a RTX-1701 capillary column and the recovered isopropyl esters derivatives were detected by flame ionization detection. The analytical method was validated for selectivity, linearity, detection and quantification limits, recovery and intra-day and inter-day precisions. Calibration curves showed linearity in the range of 8-800 mg/L, except for HA and PAUA (40-800 mg/L). Limits of detection were between 0.2 (PPA) and 7.0 (PAUA) mg/L. The intra-day precisions determined at three concentrations levels were less than 5% for BA, PAA, MA and PGA and 9% for HA and PAUA, respectively. The corresponding mean inter-day precisions for these two groups were 8 and 16%, respectively. The method was successfully applied to quantitatively analyze styrene, styrene-oxide, ethylbenzene and toluene metabolites in urine samples from rats exposed by inhalation to these compounds at levels close to the occupational threshold limit values. Provided that this method can be transposed to human urine, it could have applications as part of biological monitoring for workers exposed to styrene or related compounds.

Evaluation of tri-steps modified styrene-butadiene-styrene block copolymer membrane for wound dressing.

Tri-steps modified styrene-butadiene-styrene block copolymer (SBS) membrane was prepared with epoxidation, ring opening reaction with maleated ionomer and layer-by-layer assembled polyelectrolyte technique. The tri-steps modified SBS membrane was characterized by infrared spectroscopy and X-ray photoelectron spectroscope (XPS). The structures of the modified SBS membranes were identified with methylene blue and azocarmine G. The content of amino group on the surface of the modified membrane was calculated from uptake of an acid dye. The values of the contact angle, water absorption, water vapor transmission rate and the adsorption of fibronectin on the membranes were determined. To evaluate the biocompatibility of the tri-steps modified SBS membrane, the cytotoxicity, antibacterial and growth profile of the cell culture of 3T3 fibroblasts on the membrane were evaluated. The bactericidal activity was found on the modified SBS. From the cell culture of 3T3 fibroblasts on the membrane, it revealed that the cells not only remained viable but also proliferated on the surface of the tri-steps modified SBS membranes. As the membranes are sterile semipermeable with bactericidal activity and transparent allowing wound checks, they can be considered for shallow wound with low exudates.

Impact of noise or styrene exposure on the kinetics of presbycusis.

Presbycusis, or age-related hearing loss is a growing problem as the general population ages. In this longitudinal study, the influence of noise or styrene exposure on presbycusis was investigated in Brown Norway rats. Animals were exposed at 6 months of age, either to a band noise centered at 8 kHz at a Lex,8h = 85 dB (86.2 dB SPL for 6 h), or to 300 ppm of styrene for 6 h per day, five days per week, for four weeks. Cubic distortion product otoacoustic emissions (2f1-f2 DPOAEs) were used to test the capacity of the auditory receptor over the lifespan of the animals. 2f1-f2DPOAE measurements are easy to implement and efficiently track the age-related deterioration of mid- and high-frequencies. They are good indicators of temporary auditory threshold shift, especially with a level of primaries close to 60 dB SPL. Post-exposure hearing defects are best identified using moderate, rather than high, levels of primaries. Like many aging humans, aging rats lose sensitivity to high-frequencies faster than to medium-frequencies. Although the results obtained with the styrene exposure were not entirely conclusive, histopathological data showed the presbycusis process to be enhanced. Noise-exposed rats exhibit a loss of spiral ganglion cells from 12 months and a 7 dB drop in 2f1-f2DPOAEs at 24 months, indicating that even moderate-intensity noise can accelerate the presbycusis process. Even though the results obtained with the styrene exposure are less conclusive, the histopathological data show an enhancement of the presbycusis process.

Metabolism and toxicity of styrene in microsomal epoxide hydrolase-deficient mice.

Styrene, which is widely used in manufacturing, is both acutely and chronically toxic to mice. Styrene is metabolized by cytochromes P-450 to the toxic metabolite styrene oxide, which is detoxified via hydrolysis with microsomal epoxide hydrolase (mEH) playing a major role. The purpose of these studies was to characterize the importance of this pathway by determining the hepatotoxicity and pneumotoxicity of styrene in wild-type and mEH-deficient (mEH(-/-)) mice. While the mEH(-/-) mice metabolized styrene to styrene oxide at the same rate as the wild-type mice, as expected there was minimal metabolism of styrene oxide to glycol. mEH(-/-) mice were more susceptible to the lethal effects of styrene. Twenty-four hours following the administration of 200 mg/kg ip styrene, mice demonstrated a greater hepatotoxic response due to styrene, as measured by increased serum sorbitol dehydrogenase activity and greater pneumotoxicity as shown by increased protein levels, cell numbers, and lactate dehydrogenase activity in bronchioalveolar lavage fluid. mEH(-/-) mice were also more susceptible to styrene-induced oxidative stress, as indicated by greater decreases in hepatic glutathione levels 3 h after styrene. Styrene oxide at a dose of 150 mg/kg did not produce hepatotoxicity in either wild-type or mEH(-/-) mice. However, styrene oxide produced pneumotoxicity that was similar in the two strains. Thus, mEH plays an important role in the detoxification of styrene but not for exogenously administered styrene oxide.

Indicators of oxidative stress and apoptosis in mouse whole lung and Clara cells following exposure to styrene and its metabolites.

In mice, styrene is hepatotoxic, pneumotoxic, and causes lung tumors. One explanation for the mechanism of toxicity is oxidative stress/damage. Previous studies have shown decreased glutathione levels, linked to increased apoptosis, in lung homogenates and isolated Clara cells 3 h following styrene or styrene oxide (SO) administration or in vitro exposure. The objective of the current studies was to determine what effects styrene and its active metabolites, primarily styrene oxide, had on indicators of oxidative stress and attendant apoptosis in order to understand better the mechanism of styrene-induced toxicity. Three hours following in vitro exposure of Clara cells to styrene or SO there were increases in reactive oxygen species (ROS). Following administration of styrene or styrene oxide ip, increases in ROS, superoxide dismutase (SOD), and 8-hydroxydeoxyguanosine (8-OHdG) formation were observed. Since increases in ROS have been linked to increases in apoptosis ratios of bax/bcl-2, mRNA and protein expression were determined 3-240 h following the administration of styrene and R-styrene oxide (RSO). The bax/bcl-2 mRNA ratio increased 12 and 24 h following R-SO and 120 h following styrene administration. However, the bax/bcl-2 protein ratio was not increased until 240 h following R-SO, and 24 and 240 h following styrene administration. However, only a slight increase in caspase 3 was observed. These results indicated that oxidative stress occurred 3h following styrene or styrene oxide as evidenced by increased ROS and SOD. This increased ROS may be responsible for the increased 8-OHdG formation. Our findings of limited apoptosis in Clara cells following acute exposure to styrene or SO are in agreement with others and may reflect the minimal extent to which apoptosis plays a role in acute styrene toxicity. It is clear, however, that oxidative stress and oxidative effects on DNA are increased following exposure to styrene or styrene oxide, and these may play a role in the lung tumorigenesis in mice.

Investigation on the mechanisms of genotoxicity of butadiene, styrene and their combination in human lymphocytes using the Comet assay.

The toxicity of butadiene and styrene is exerted by their metabolites. Such metabolites have been extensively scrutinized at the in vitro level demonstrating evident genotoxic properties. In monitoring, a diverse range of outcomes has been produced. Additionally, epidemiological studies in rubber workers face difficulties of data interpretation due to the changeability and multiple exposures of the workers as well as to confounding factors inherent to the cohorts. Nevertheless, toxicity has been associated with a significant trend of increasing the risk of leukaemia in employees at the styrene-butadiene rubber industry. Thus, further effort must be made to distinguish the exposures to each chemical over time and to characterize their interrelationships. The present investigation focuses on the effects and mechanisms of damage of the mixture styrene-butadiene by examining its metabolites: styrene oxide (SO), butadiene monoepoxide (BME) and butadiene diepoxide (BDE) respectively. The in vitro Comet assay on frozen lymphocytes has been employed to ascertain the DNA damage patterns for the styrene-butadiene metabolites combined and on their own. Different patterns were observed for the mixture and each of its components. This study has also led to determining the mechanism of damage of the mixture and the compounds. With regard to the presence of reactive oxygen species (ROS), co-treatment with catalase does not modulate the genotoxicity of the mixture but it does modulate its components. The outcomes also indicate that the mixture induces cross-links and this is due to the influence of BDE in the mixture, being more evident as the concentration of BDE increases. An investigation on the sensitivity of lymphocytes from occupationally un/exposed subjects to in vitro exposure of the mixture and its components revealed that occupationally exposed subjects had a substantially higher background of DNA damage and a lower sensitivity to the metabolites of styrene, 1,3-butadiene and its mixture.

Styrene maleic acid-pirarubicin disrupts tumor microcirculation and enhances the permeability of colorectal liver metastases.

BACKGROUND: Doxorubicin is a commonly used chemotherapy limited by cardiotoxicity. Pirarubicin, derived from doxorubicin, selectively targets tumors when encapsulated in styrene maleic acid (SMA), forming the macromolecular SMA pirarubicin. Selective targeting is achieved because of the enhanced permeability and retention (EPR) effect. SMA-pirarubicin inhibits the growth of colorectal liver metastases, but tumor destruction is incomplete. The role played by the tumor microcirculation is uncertain. This study investigates the pattern of microcirculatory changes following SMA-pirarubicin treatment. METHODS: Liver metastases were induced in CBA mice using a murine-derived colon cancer line. SMA-pirarubicin (100 mg/kg total dose) was administered intravenously in 3 separate doses. Twenty-four hours after chemotherapy, the tumor microvasculature was examined using CD34 immunohistochemistry and scanning electron microscopy. Tumor perfusion and permeability were assessed using confocal in vivo microscopy and the Evans blue method. RESULTS: SMA-pirarubicin reduced the microvascular index by 40%. Vascular occlusion and necrosis were extensive following treatment. Viable cells were arranged around tumor vessels. Tumor permeability was also increased. CONCLUSION: SMA-pirarubicin damages tumor cells and the tumor microvasculature and enhances tumor vessel permeability. However, tumor necrosis is incomplete, and the growth of residual cells is sustained by a microvascular network. Combined therapy with a vascular targeting agent may affect residual cells, allowing more extensive destruction of tumors.

Excretion of urinary N7 guanine and N3 adenine DNA adducts in mice after inhalation of styrene.

New urinary adenine adducts, 3-(2-hydroxy-1-phenylethyl)adenine (N3alphaA), 3-(2-hydroxy-2-phenylethyl)adenine (N3betaA), were found in the urine of mice exposed to styrene vapour. These styrene 7,8-oxide derived adenine adducts as well as previously identified guanine adducts, 7-(2-hydroxy-1-phenylethyl)guanine (N7alphaG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7betaG) were quantified by HPLC-ESI-MS(2) and the excretion profile during and after a repeated exposure to 600mg/m(3) or 1200mg/m(3) of styrene for 10 consecutive days (6h/day) was determined. The excretion was dose dependent. Total N3 adenine adducts (N3alphaA+N3betaA) excreted amounted to nearly 0.8x10(-5)% of the absorbed dose while urinary N7 guanine adducts (N7alphaG+N7betaG) amounted to nearly 1.4x10(-5)% of the dose. No accumulation of the adducts was observed. Due to rapid depurination from the DNA, the excretion of both N3 adenine and N7 guanine adducts ceased shortly after finishing the exposure. Both N3 adenine and N7 guanine adducts may be used as non-invasive biomarkers of effective dose reflecting only a short time exposure to styrene.

Relation between outer hair cell loss and hearing loss in rats exposed to styrene.

The relationship between outer hair cell (OHC) loss and cochlear sensitivity is still unclear, because in many animal models there exist surviving but dysfunctional OHCs and also injured/dead inner hair cells (IHC). Styrene is an ototoxic agent, which targets and destroys OHCs starting from the third row to the second and first rows depending on the exposure level. The remaining cells may be less affected. In this experiment, rats were exposed to styrene by gavage at different doses (200-800 mg/kg/day) for varying periods (5 days/week for 3-12 weeks). An interesting finding was that the cochlear sensitivity was not affected in a few rats with all OHCs in the third row being destroyed by styrene. A further loss of OHCs was usually accompanied with a linear input/output (I/O) function of cochlear compound action potentials (CAP), indicating the loss of cochlear amplification. However, normal CAP amplitudes at the highest stimulation level of 90 dB SPL were often observed when all OHCs were destroyed, indicating normal function of the remaining IHCs. The OHC-loss/hearing-loss relation appeared to be a sigmoid-type function. Initially, styrene-induced OHC losses (<33%) did not result in a significant threshold shift. Then CAP threshold shift increased dramatically with OHC loss from 33% to 66%. Then, CAP threshold changed less with OHC loss. The data suggest a tri-modal relationship between OHC loss and cochlear amplification. That is, under the condition that all surviving OHCs are ideally functioning, the cochlear amplifier is not affected until 33% of OHCs are absent, then the gain of the amplifier decreases proportionally with the OHC loss, and at last the amplifier may fail completely when more than 67% of OHCs are lost.

Styrene induces an inflammatory response in human lung epithelial cells via oxidative stress and NF-kappaB activation.

Styrene is a volatile organic compound (VOC) that is widely used as a solvent in many industrial settings. Chronic exposure to styrene can result in irritation of the mucosa of the upper respiratory tract. Contact of styrene with epithelial cells stimulates the expression of a variety of inflammatory mediators, including the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). To characterise the underlying mechanisms of the induction of inflammatory signals by styrene, we investigated the influence of this compound on the induction of oxidative stress and the activation of the nuclear factor-kappa B (NF-kappaB) signalling pathway in human lung epithelial cells (A549). The results demonstrate that styrene-induced MCP-1 expression, as well as the expression of the oxidative stress marker glutathione S-transferase (GST), is associated with a concentration dependent pattern of NF-kappaB activity. An inhibitor of NF-kappaB, IKK-NBD, and the anti-inflammatory antioxidant N-acetylcysteine (NAC) were both effective in suppressing styrene-induced MCP-1 secretion. In addition, NAC was capable of inhibiting the upregulation of GST expression. Our findings suggest that the activation of the NF-kappaB signalling pathway by styrene is mediated via a redox-sensitive mechanism.