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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Differential gene expression by styrene in rat reproductive tissue.

Styrene is an important industrial chemical that is extensively used in the production of resins, rubbers and fiberglass-reinforced plastics. Exposing male rats to high doses of styrene may produce sperm abnormalities or infertility. To determine the mechanism underlying styrene-mediated toxicity in male reproductive organs, a reverse transcription-polymerase chain reaction (RT-PCR) technology was employed using annealing control primers (ACPs) to identify the differentially expressed genes following styrene treatment in isolated testis of male rats. By using 120 ACPs, a total of 6 expressed sequence tags (ESTs) of genes were differentially expressed in styrene-treated rats, as compared to untreated, which were cloned and sequenced. Of the genes analyzed, 5 genes (testis-specific expressed gene 101, protein kinase C, H+-ATPase isoform 2, peroxiredoxin 1, and aquaporin 9) were inducible and one gene expression (clusterin) was significantly suppressed by styrene. Regulation of each gene by styrene was confirmed by RT-PCR. It was shown that styrene decreased clusterin expression in a concentration-dependent manner and these effects occurred mainly in testis. Taken together, these results indicate that repression of clusterin gene expression by styrene may play an important role in styrene-mediated toxicities.

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.

Inhaled chemicals may enhance allergic airway inflammation in ovalbumin-sensitised mice.

Occupational allergy and asthma is a challenging issue in the developing countries. Chemicals inhaled in the workplaces may act not only as allergens but also as immune response modifiers, contributing to asthma exacerbation. In this study, we tested the adjuvant effect of 20 ppm chloroform, 10 ppm 1,1-dichloroethylene, and 100 ppm styrene in mice. Female BALB/c mice were sensitised to ovalbumin (OVA) without using alum. During the OVA-sensitisation period, these mice were exposed by inhalation to the chemicals studied for 6h/day for four consecutive days. After two OVA-intratracheal challenges, a mild Th2 immune response was observed in the OVA-exposed groups. This response was characterised by a mild increase in serum specific IgE level, in local Th2 cytokine production, and in lung inflammatory reaction. Exposure to styrene or chloroform alone slightly increased Th2 cytokine production by lung-draining lymph node cells cultured with concanavaline A, except for the IL-4 level in the chloroform exposure group, which decreased. On the other hand, exposure to 1,1-dichloroethylene alone markedly increased the Th2 cytokine levels compared to those observed in the groups exposed to OVA alone. In the combined OVA+chemical-treated groups, styrene potentiated IL-4, -5 and -13 production efficiently (approximately two, four and three times higher, respectively), resulting in an increase in the total IgE levels and inflammatory reaction. On the other hand, the enhanced IgE levels and the exacerbation of the inflammatory response by 1,1-dichloroethylene or chloroform were associated with only minor changes in local cytokine levels. These findings suggest that exposure to chemicals through inhalation may aggravate the allergic lung inflammation. And this, depending on the chemical exposure conditions, may result from the synergistic effect of chemicals and allergen on local Th2 cytokine production.

Styrene hepatotoxicity in rats treated by inhalation or intraperitoneally: a structural investigation.

The purpose of this study was to investigate the toxicity of styrene in the liver of adult rats treated either by inhalation of styrene vapour (300 ppm, 6 h/d, 5 d/wk, for 2 wk) or intraperitoneally with different styrene doses (4, 40, 400 mg/Kg) for 3 consecutive days. Using a light microscope, some alterations of liver parenchyma and sinusoid dilation were noticed, more marked in the group treated with the intraperitoneal administration of the chemical. Using an electron microscope, some additional changes were observed (once again, more marked in the latter group of rats): a) an increase in the content of lipids inside hepatocytes, and b) the rise of intracytoplasmic, intercellular and perisinusoidal collagen fibres. Therefore, cell damage and functional disturbance of sinusoids due to perisinusoidal fibrosis are apparent in the liver of both groups of rats exposed to styrene treatment, but these changes are definitely more significant in those subjected to intraperitoneal administration.

The role of cytochromes P-450 in styrene induced pulmonary toxicity and carcinogenicity.

Exposure of CD-1 mice to atmospheres of 40 and 160 ppm styrene, daily for up to 10 days, caused pulmonary toxicity characterised by focal loss of cytoplasm and focal crowding of non-ciliated Clara cells, particularly in the terminal bronchiolar region. The toxicity was accompanied by an increase in cell replication rates in terminal and large bronchioles of mice exposed for 3 days or longer. The toxicity and increased cell replication were no longer apparent after a 2-day break in exposure, but re-occurred when exposure was resumed. Similar effects were seen in mice given oral doses of 10, 100 or 200 mg/kg styrene, daily for 5 days. Increases in cell replication rates were seen in the terminal bronchioles in mice dosed with 100 and 200 mg/kg styrene, but not 10 mg/kg. Toxicity was limited to 3 to 10 animals in the 200 mg/kg group. Neither morphological nor cell proliferation effects were seen in the alveolar region of the mouse lung in any of these studies, nor were any effects observed in the lungs of CD rats exposed to 500 ppm styrene for up to 10 days. The pulmonary toxicity and increased cell division seen in mice, but not rats, correlates with the known species differences in pulmonary carcinogenicity of styrene, suggesting that the acute and chronic responses are causally related. 5-Phenyl-1-pentyne was shown to inhibit the pulmonary cytochrome P-450 metabolism of styrene in vivo. Cell replication rates in the lungs of mice treated with this inhibitor and exposed to styrene were comparable with controls demonstrating that the pulmonary effects of styrene on the mouse lung are caused by a metabolite of styrene, probably styrene oxide. The risks associated with exposure to styrene appear to correlate well with the metabolic capacity of the lung.

Effect of inhaled industrial chemicals on systemic and local immune response.

Using immunotoxic functional tests, namely IgM response to sheep red blood cells (SRBCs) and interferon-gamma (IFN-gamma) production, this study simultaneously evaluated the effects of inhaled chloroform (10, 20, and 50 ppm), carbon tetrachloride (100, 200, and 300 ppm), 1,1-dichloroethylene (5, 10, and 15 ppm), and styrene (100, 200, and 300 ppm) on the systemic (spleen) and local (lung-associated lymph nodes) immune response. At least one concentration of all the chemicals studied provoked a statistically significant increase in IgM response in the lymph nodes compared with the controls, as expressed by the number of plaque-forming cells (PFCs), whereas only the highest concentration of 1,1-dichloroethylene provoked an increase in the number of PFCs statistically different from the controls in the case of the spleens. The release of IFN-gamma in the lymph node cell cultures of the exposed mice exceeded that of the controls by more than 600%, whereas the release of IFN-gamma in the spleen cell cultures of the exposed mice was moderately different from the controls. It would appear from these results that the lung-associated lymph nodes are sensitive targets for chemical inhalation and that the results of systemic tests in the spleen may not mirror local immune response dysfunction. For risk assessment of inhaled chemicals, it is therefore important to take the local immunotoxic effects into consideration, in particular immunostimulation which may be involved in the rise in allergic diseases in industrialised countries.

Dose-response implications of the University of Alabama study of lymphohematopoietic cancer among workers exposed to 1,3-butadiene and styrene in the synthetic rubber industry.

New quantitative cancer risk estimates for exposure to 1,3-butadiene are presented. These estimates are based on the most recent human epidemiologic data developed by Drs Delzell and Macaluso and their colleagues at the University of Alabama at Birmingham. The implications of Poisson regression analyses of the relative rate for leukemia are explored using their updated dose estimates and lymphohematopoietic cancer data. The Poisson regression model in these analyses has the same form as in the U.S. Environmental Protection Agency (EPA)'s draft risk assessment of 1,3-butadiene [U.S. Environmental Protection Agency, Health Risk Assessment of 1,3-Butadiene - External Review Draft, National Center for Environmental Assessment, Office of Research and Development, 63 Fed. Reg. 7167 (February 12, 1998) Publication NCEA-W-0267, Washington, 1998]. Consistent with the proposed cancer risk assessment guidelines of the EPA and the EPA's draft risk assessment, the exploration includes the maximum likelihood estimate of the 'effective concentration' (EC(01)) corresponding to an extra risk of leukemia of 0.01 (1%) from a lifetime continuous exposure to 1,3-butadiene based on a linear dose-response model and the cumulative 1,3-butadiene dose metric (ppm-years). The incorporation of the most recent exposure estimates results in a 2.5-fold decrease in the estimates of leukemia risks computed by EPA. In addition, three changes proposed by the American Chemistry Council (formerly the Chemical Manufacturers Association) to the EPA's Science Advisory Board (SAB) for EPA's draft risk assessment of 1,3-butadiene are incorporated into the calculation. This results in approximately an additional fivefold decrease in the risk estimates of leukemia. The leukemia cancer risk estimates in the EPA's draft risk assessment of 1,3-butadiene decrease by approximately a factor of 13-fold when the updated epidemiologic data and the alternative numbers proposed by industry to the SAB are both incorporated. Specifically, the maximum likelihood estimate of the EC(01) increases from EPA's 1.2 ppm to 2.8 ppm on the basis of the updated epidemiologic data and increases further to 15.1 ppm when the CMA's proposed changes are also incorporated.