Enhanced oxidative stress in the skin of vitamin E deficient mice exposed to semisynthetic metal working fluids

Metal working fluids (MWFs) are widely used in industry for metal cutting, drilling, shaping, lubricating, and milling. Many occupational health concerns have arisen for workers exposed to MWFs. It has been reported earlier that occupational exposure to MWFs causes allergic and irritant contact dermatitis. Previously, we have shown that dermal exposure of female and male B6C3F1 mice to 5% MWFs for 3 months resulted in accumulation of mast cells and elevation of histamine in the skin. Topical exposure to MWFs also resulted in elevated oxidative stress in the liver of both sexes and the testes in males. The goal of this study was to evaluate whether preexisting oxidative stress in the skin exacerbated mast cell influx after MWFs treatment. Oxidative stress in the skin of B6C3F1 mice was generated by dietary vitamin E deprivation. Mice were given vitamin E deficient (5-10 i.v./kg of vitamin E) or basal (50 i.v./kg of vitamin E) diets for 34 weeks. Topical treatment with MWFs (100 microl, 30%) started after 18 weeks of alimentary vitamin E deprivation. Histology of the skin after 16 weeks of exposure to MWFs revealed a 53% increase in mast cell accumulation in vitamin E deficient diets compared to mice given a vitamin E sufficient diet. Total antioxidant reserve in skin of vitamin E deprived mice treated with MWFs was decreased by 66% as compared to those mice given a vitamin E sufficient diet. GSH and protein thiols in the dermis of vitamin E deprived mice exposed to MWFs were also decreased 39 and 42%, respectively, as compared to mice given basal diet. This study clearly delineates the role of oxidative stress in enhancing mast cell accumulation caused by topical exposure to MWFs.     

Dietary antioxidants and the biochemical response to oxidant inhalation: I. Influence of dietary vitamin E on the biochemical effects of nitrogen dioxide exposure in rat lung

Sprague-Dawley rats derived from a specific pathogen-free colony were raised from birth on a test diet containing either 0 or 50 IU vitamin E/kg diet for 8 weeks. Rats from each dietary group were exposed to 3 ppm (5640 μg/m³) nitrogen dioxide (NO₂) continuously for 7 days. They were then killed, and the lungs analyzed for changes in weight, DNA and protein contents, tissue oxygen utilization, sulfhydryl metabolism, and the activities of NADP-reducing enzymes. The difference in dietary vitamin E alone did not cause any significant changes in these parameters. However, after NO₂ exposure the changes in these parameters relative to their corresponding unexposed controls were greater for the deficient rats than for the supplemented rats. The biochemical changes observed may be a response of the lung to injury from NO₂ exposure. The larger changes in the lungs of deficient rats may reflect a greater sensitivity of these animals to inhaled NO₂. The vitamin E contents of lung tissue in deficient and supplemented rats reflected the dietary levels. After NO₂ exposure, the vitamin E content in the lung increased significantly in supplemented rats but decreased in the deficient rats relative to their corresponding unexposed controls. The elevation of vitamin E levels in the lungs of supplemented rats with NO₂ exposure suggests its mobilization from other body sites, whereas in deficient rats this process may not have been possible.     

Inflammatory and immunological responses to subchronic exposure to endotoxin-contaminated metalworking fluid aerosols in F344 rats

Rats were exposed for 6 h per day in inhalation chambers to a 10 mg/m(3) concentration of metalworking fluid (MWF) contaminated with endotoxin at concentrations of 1813 (low dose) and 20,250 eu/m(3) (high dose) 5 days per week for 8 weeks. It was found that 94.7% of the MWF aerosol particles had diameters in the range of 0.42-4.6 microm, with geometric mean diameter of 1.56 microm. The body weight and pulmonary function parameters were measured every week during the 8 weeks of exposure, whereas bronchoalveolar lavage (BAL) fluid was prepared to measure the inflammatory markers and cytokines after the 8 weeks of exposure. There were no changes in body weight and respiratory function (tidal volume and respiratory frequency) during the 8 weeks of exposure to the MWF containing endotoxins, yet lung weight increased significantly (P < 0.05) in the rats exposed to the MWF both with and without endotoxins. The number of polymorphonuclear (PMN) cells in the BAL fluid increased significantly (P < 0.05) in the rats exposed to MWF with endotoxins, and the levels of cytokines such as IL-4, INF-gamma, IL-1beta, and TNF-alpha also were significantly increased (P < 0.05) compared to the control. The NOx production activity of the BAL cells increased significantly (P < 0.05) in the rats exposed to the MWF with and without endotoxins. Increases in lung weight, number of PMN cells, and levels of extracellular cytokines and NOx were all more significant in the rats exposed to the MWF with endotoxins rather than in those exposed to MWF without endotoxins. In spleen cell cultures, T-cell proliferation activity was decreased, yet cytokine levels (INF-gamma, IL-1beta, IL-4, and TNF-alpha) remained unchanged after repeated exposure to MWF with and without endotoxins. Although the levels of total IgG(1), IgG(2a), and IgE antibodies in the serum were not changed, the levels of endotoxin-specific antibodies, including IgG(2a) and IgE, were increased significantly (P < 0.05) in the rats exposed to endotoxins, but there was not a significant increase in endotoxin-specific IgG(1). When taken together, the results indicate that lung inflammatory responses can be induced without changing pulmonary function after repeated exposure to MWFs contaminated with endotoxins. In addition, endotoxin-specific IgG(2a) and IgE may be effective biomarkers for workers exposed to MWFs contaminated with endotoxins in the workplace.     

DERMAL AND SYSTEMIC TOXICITY AFTER APPLICATION OF SEMISYNTHETIC METAL-WORKING FLUIDS IN B6C3F1 MICE

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 µl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 5% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5% MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

Dermal and systemic toxicity after application of semisynthetic metal-working fluids in B6C3F1 mice

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 microl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 55% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5%, MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

An approach for evaluating the respiratory irritation of mixtures: application to metalworking fluids

 Recently, the sensory and pulmonary irritating properties of ten metalworking fluids (MWF) were assessed using a mouse bioassay. Relative potency of the MWFs was estimated, but it was not possible to identify the component(s) responsible for the the respiratory irritation induced by each MWF. One of the ten fluids, MWF “E”, produced sensory and pulmonary irritation in mice, and it was of moderate potency in comparison to the other nine MWFs. MWF “E” had three major components: tall oil fatty acids (TOFA), sodium sulfonate (SA), and paraffinic oil (PO). In the present study, the sensory and pulmonary irritating properties of these individual components of MWF “E” were evaluated. Mixtures of the three components were also prepared and similarly evaluated. This analysis revealed that the sensory irritation from MWF “E” was largely due to TOFA, whereas SA produced the pulmonary irritation observed with MWF “E”. Both TOFA and SA were more potent irritants than was MWF “E”, and the potency of TOFA and/or SA was diminished through combination with PO. There was no evidence of synergism of the components when combined to form MWF “E”. This approach for identifying the biologically “active” component(s) in a mixture should be useful for other MWFs. Furthermore, the approach should be easily adapted for other applications involving concerns with mixtures. Received: 2 November 1994/Accepted: 16 March 1995     

Skin absorption of six performance amines used in metalworking fluids

Every year, 10 million workers are exposed to metalworking fluids (MWFs) that may be toxic. There are four types of MWFs: neat oils and three water‐based MWFs (soluble oil, semisynthetic and synthetic), which are diluted with water and whose composition varies according to the mineral oils ratio. MWFs also contain various additives. To determine the absorption of six amines used as corrosion inhibitors and biocides in MWFs, porcine skin flow‐through diffusion cell experiments were conducted with hydrophilic ethanolamines (mono‐, di‐ and triethanolamine, MEA, DEA and TEA respectively) and a mixture of lipophilic amines (dibutylethanolamine, dicyclohexylamine and diphenylamine). The six amines were dosed in four vehicles (water and three generic water‐based MWF formulations) and analyzed using a scintillation counter or gas chromatography/mass spectrometry. These 24 h studies showed that dermal absorption significantly (P < 0.05) increased from water for the six amines (e.g. 1.15 ± 0.29% dose; DEA in water) compared to other formulations (e.g. 0.13 ± 0.01% dose; DEA in semisynthetic MWF) and absorption was greatest for dibutylethanolamine in all the formulations. The soluble oil formulation tended to increase the dermal absorption of the hydrophilic amines. The permeability coefficient was significantly higher (P < 0.05) with TEA relative to the other hydrophilic amines (e.g. 4.22 × 10^–4 ± 0.53 × 10^–4 cm h^–1 [TEA in synthetic MWF] vs. 1.23 × 10^–4 ± 0.10 × 10^–4 cm h^–1 [MEA in synthetic MWF]), except for MEA in soluble oil formulation. Future research will confirm these findings in an in vivo pig model along with dermatotoxicity studies. These results should help MWF industries choose safer additives for their formulations to protect the health of metalworkers. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of sulfite on macrophage functions of normal and sulfite oxidase-deficient rats.

Sulfite has both an endogenous and an exogenous provenance in the mammalian tissues. The aim of the present study was to assess the effect of sulfite on macrophages functions in normal or sulfite oxidase deficient rats. Rats were divided into eight groups; (1) control group, (2) sulfite group (the rats received sodium meta bi-sulfite (25 mg/kg) in drinking water for 6 weeks), (3) vitamin E group (the rats received Vit E (50 mg/kg) by gavage for 6 weeks), (4) sulfite group+Vit E, (5)sulfite oxidase deficient group (the rats received high-W/Mo-deficient diet. The activity of sulfite oxidase was reduced in rats maintained on the high-W/Mo-deficient diet during the first 21 days of treatment. After the sulfite-oxidase deficiency, the rats continued to receive high-W/Mo-deficient diet for 6 weeks.), (6) sulfite+sulfite oxidase deficient group, (7) Vit E+sulfite oxidase deficient group, and (8) sulfite+Vit E+sulfite oxidase deficient group. Sulfite caused a significant increase in phagocytic and chemotactic activities of peritoneal macrophages. In sulfite-oxidase deficient rats, the increase in phagocytic and chemotactic activities in peritoneal macrophages after sulfite intake was found more than the control rats. Vit E supplementation prevented sulfite induced increase in macrophages functions. These results show that the macrophage functions are sensitive to sulfite intake. The effect of sulfite on macrophage functions may be related to reactive oxygen species. Because Vit E administration was able to modulate significantly sulfite-induced changes in the functions of peritoneal macrophages.     

Respiratory effect of acute and subacute exposure to endotoxin-contaminated metal working fluid (MWF) aerosols on Sprague-Dawley rats

Male Sprague-Dawley rats were exposed to a water-soluble metal working fluid (MWF) (5% v/v) contaminated with endotoxins (10,000 eu/ml or 100,000 eu/ml) at 10 mg/m³ for six hours per day for three days (acute exposure) or two weeks (subacute exposure). The geometric mean diameter of the MWF aerosols was 1.56 m, and the airborne endotoxin concentrations ranged from 1,231 to 2,173 eu/m³ (10,000 eu/ml in the bulk MWF) for the low dose and 19,263–27,386 eu/m³ (100,000 eu/ml in the bulk MWF) for the high dose. Minimal effects were observed after exposure to 10 mg/m³ of the MWF without endotoxins for three days or two weeks. However, an increase in the number of polymorphonuclear cells (PMNs) and the level of protein was noted in the bronchoalveolar lavage (BAL) fluid from the rats acutely exposed to the MWF with endotoxins. The acute exposure produced a greater increase in the number of PMNs and total cell number in the BAL fluid than the subacute exposure. The number of white blood cells in the peripheral blood and the weight of the lungs both increased after the subacute exposure to the MWF aerosol with endotoxins, indicating increased vascular permeability in response to the endotoxin exposure. The levels of cyotokines such as IL-4, INF-, and IL-1 in the BAL fluid from the rats exposed to the MWF with or without endotoxins remained unchanged. Although the level of nitric oxide (NO_x) in the BAL supernatant did not show any change, the induction of NO_x from the alveolar macrophages increased in the rats acutely or subacutely exposed to the MWF contaminated with endotoxins. The ConA-induced proliferation response showed no change, yet the LPS-induced proliferation response was significantly increased in the splenocytes from the rats subacutely exposed to the MWF with and without endotoxins. The level of TNF- in the spleen cell culture obtained from the rats exposed to the MWF with or without endotoxins increased without changing the levels of IL-1, IL-4, and INF-. The level of endotoxin-specific IgE in the serum obtained from the rats exposed to the MWF with endotoxins increased dose-dependently, while the levels of total immunoglobulins (IgG₁, IgG_2a and IgE) and endotoxin-specific IgG₁ and IgG_2a remained unchanged. Accordingly, the current results indicate that lung inflammation can be immediately induced by acute or subacute exposure to an MWF contaminated with endotoxins, and macrophages would appear to play a role in the induction of inflammation along with B-cell functions rather than T-cell functions, after subacute exposure to an MWF with endotoxins. In addition, endotoxin-specific IgE is an early marker for endotoxin exposure in the workplace.     

Methotrexate use in rheumatoid arthritis. A Clinician's perspective

Aminopterine, a precursor of methotrexate (MTX), was first used for the treatment of rheumatoid arthritis (RA) in 1951 [Gubner, R., 1951. Therapeutic suppression of tissue reactivity: I. Comparison of the effects of cortisone and aminopterin. Am. J. Med. Sci. 221, 169-175; Gubner, R., August, S., Ginsberg, V., 1951. Therapeutic suppression of tissue reactivity: II. Effect of aminopterin in rheumatoid arthritis and psoriasis. Am. J. Med. Sci. 221, 176-182.]. Corticosteroids, and to some extent cyclophosphamide, took MTX out of the rheumatologist's armamentarium until the late 1970s-early 1980s when the toxic profile of these compounds became apparent. By the mid 1980s, four randomized clinical trials (RCTs) had proven beyond doubt the beneficial effects of MTX when administered to patients with established disease who had failed to respond to other compounds such as gold salts and D-penicillamine [Thompson, R.N., Watts, C., Edelman, J., Esdaile, J., and Russell, A.S., 1984. A controlled two-centre trial of parenteral methotrexate therapy for refractory rheumatoid arthritis. J. Rheumatol. 11, 760-763; Andersen, P.A., West, S.G., O'Dell, J.R., Via, C.S., Claypool, R.G., and Kotzin, B.L., 1985. Weekly pulse methotrexate in rheumatoid arthritis. Clinical and immunologic effects in a randomized, double-blind study. Ann. Intern. Med. 103, 489-496; Weinblatt, M.E., Coblyn, J.S., Fox, D.A., Fraser, P.A., Holdsworth, D.E., Glass, D.N., and Trentham, D.E., 1985. Efficacy of low-dose methotrexate in rheumatoid arthritis. N. Engl. J. Med. 312, 818-822; Williams, H.J., Willkens, R.F., Samuelson, C.O.J., Alarcón, G.S., Guttadauria, M., Yarboro, C., Polisson, R.P., Weiner, S.R., Luggen, M.E., Billingsley, L.M., Dahl, S.L., Egger, M.J., Reading, J.C., and Ward, J.R., 1985. Comparison of low-dose oral pulse methotrexate and placebo in the treatment of rheumatoid arthritis. A controlled clinical trial. Arthritis Rheum. 28, 721-730.]. Subsequently, these four trials were included in a meta-analysis and the drug was approved by the Food and Drug Administration for use in RA [Health and Public Policy Committee, H.P.P.C. and American College Physicians, A.C.P., 1987. Methotrexate in rheumatoid arthritis. Ann. Intern. Med. 107, 418-419; Paulus, H.E., 1986. FDA Arthritis Advisory Committee meeting: Methotrexate; guidelines for the clinical evaluation of antiinflammatory drugs; DMSO in scleroderma. Arthritis Rheum. 29, 1289-1290; Tugwell, P., Bennett, K., and Gent, M., 1987. Methotrexate in rheumatoid arthritis. Indications, contraindications, efficacy, and safety. Ann. Intern. Med. 107, 358-366.]. Since then, rheumatologists have become aware of what Pincus et al. have called "the side effects" of RA comparing the morbidity and mortality caused by RA with that potentially caused by medications used to treat this disease [Pincus, T. and Callahan, L.F., 1993. The "side effects" of rheumatoid arthritis: joint destruction, disability and early mortality. Br. J. Rheumatol. 32, 28-37.]. Thus, during the 1990s the use of MTX for the treatment of RA became generalized [O'Dell, J.R., 1997. Methotrexate use in rheumatoid arthritis. Rheum. Dis. Clin. N Am. 23, 779-796 (a); Bannwarth, B., Vernhes, J., Schaeverbeke, T., and Dehais, J., 1995. The facts about methotrexate in rheumatoid arthritis. Rev. Rhum. 62, 471-473 (b); Bologna, C., Jorgensen, C., and Sany, J., 1997a. Methotrexate as the initial second-line disease modifying agent in the treatment of rheumatoid arthritis patients. Clin. Exp. Rheumatol. 15, 597-601; Bologna, C., Viu, P. (ABSTRACT TRUNCATED)     

Application of PBPK modeling in support of the derivation of toxicity reference values for 1,1,1-trichloroethane

PBPK modeling has been increasingly applied in chemical risk assessment for dose, route, and species extrapolation. The use of PBPK modeling was explored in deriving toxicity reference values for 1,1,1-trichloroethane (1,1,1-TCE). This effort involved a 5-step process: (i) reconstruction of several published PBPK models for 1,1,1-TCE in the rat and human; (ii) selection of appropriate pharmacokinetic datasets for model comparison; (iii) determination of the most suitable PBPK model for supporting reference value derivation; (iv) PBPK model simulation of two critical studies to estimate internal dose metrics; and (v) calculation of internal dose metrics for human exposure scenarios for reference value derivation. The published model by Reitz et al. [Reitz, R.H., McDougal, J.N., Himmelstein, M.W., Nolan, R.J., Schumann, A.M., 1988. Physiologically based pharmacokinetic modeling with methylchloroform: implications for interspecies, high dose/low dose, and dose route extrapolations. Toxicol. Appl. Pharmacol. 95, 185-199] was judged the most suitable. This model has liver, fat, and rapidly and slowly perfused compartments, contains a saturable process for 1,1,1-TCE hepatic metabolism, and accommodates multiple exposure pathways in three species. Data from a human volunteer study involving acute inhalation exposure [Mackay, C.J., Campbell, L., Samuel, A.M., Alderman, K.J., Idzikowski, C., Wilson, H.K., Gompertz, D., 1987. Behavioral changes during exposure to 1,1,1-trichloroethane: time-course and relationship to blood solvent levels. Am. J. Ind. Med. 11, 223-239] and a chronic rat inhalation study [Quast, J.F., Calhoun, L.L., Frauson, L.E., 1988. 1,1,1-Trichloroethane formulation: a chronic inhalation toxicity and oncogenicity study in Fischer 344 rats and B6C3F1 mice. Fundam. Appl. Toxicol. 11, 611-625] were selected to simulate appropriate internal dosimetry data from which to derive reference value points of departure. Duration, route, and species extrapolations were performed based on internal dose metrics.     

Dietary antioxidants and the biochemical response to oxidant inhalation. II. Influence of dietary selenium on the biochemical effects of ozone exposure in mouse lung

We examined the influence of dietary selenium (Se) on the pulmonary biochemical response to ozone (O3) exposure. For 11 weeks, weanling female strain A/St mice were fed a test diet containing Se either at 0 ppm (-Se) or 1 ppm (+Se). Each diet contained 55 ppm vitamin E (vit E). Mice from each dietary group were exposed to 0.8 +/- 0.05 ppm (1568 +/- 98 micrograms/m3) O3 continuously for 5 days. After O3 exposure, they were killed along with a matched number of unexposed controls, and their lungs were analyzed for various biochemical parameters. The Se contents of lung tissue and whole blood were determined, and the levels were seven- to eightfold higher in +Se mice than in -Se mice, reflecting the Se intake of the animals. In unexposed control mice, Se deficiency caused a decline in glutathione peroxidase (GP) activity relative to +Se group. After O3 exposure, the GP activity in the -Se group was associated with a lack of stimulation of glutathione reductase (GR) activity and the pentose phosphate cycle (PPC) as assessed by measuring glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. In contrast, the +Se group after O3 exposure exhibited increases in all four enzyme activities. Other parameters, e.g., lung weight, total lung protein, DNA and nonprotein sulfhydryl contents, and O2 consumption, were not affected by dietary Se in the presence or absence of O3 exposure. The data indicate that dietary Se alters the GP activity, which in turn influences the GR and PPC activities in the lung evidently through a reduced demand for NADPH. The level of vit E in the lung was found to be twofold higher in the -Se group than in the +Se group, suggesting a compensatory relationship between Se and vit E in the lung. With O3 exposure, both Se and vit E contents further increased in the lungs of each dietary group. It is plausible that Se and vit E under oxidant stress are "mobilized" to the lung from other body sites.     

Benzene and human health: A historical review and appraisal of associations with various diseases

Over the last century, benzene has been a well-studied chemical, with some acute and chronic exposures being directly associated with observed hematologic effects in humans and animals. Chronic heavy exposures to benzene have also been associated with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) in humans. Other disease processes have also been studied, but have generally not been supported by epidemiologic studies of workers using benzene in the workplace. Within occupational cohorts with large populations and very low airborne benzene exposures (less than 0.1–1.0 ppm), it can be difficult to separate background disease incidence from those occurring due to occupational exposures. In the last few decades, some scientists and physicians have suggested that chronic exposures to various airborne concentrations of benzene may increase the risk of developing non-Hodgkin's lymphoma (NHL) (Savitz and Andrews, 1997, Am J Ind Med 31:287–295; Smith et al., 2007, Cancer Epidemiol Biomarkers Prev 16:385–391), multiple myeloma (MM) (Goldstein, 1990, Ann NY Acad Sci 609:225–230; Infante, 2006, Ann NY Acad Sci 1076:90–109), and various other hematopoietic disorders. We present a state-of-the-science review of the medical and regulatory aspects regarding the hazards of occupational exposure to benzene. We also review the available scientific and medical evidence relating to benzene and the risk of developing various disorders following specific levels of exposure. Our evaluation indicates that the only malignant hematopoietic disease that has been clearly linked to benzene exposure is AML. Information from the recent "Benzene 2009," a symposium of international experts focusing on the health effects and mechanisms of toxicity of benzene, hosted by the Technical University of Munich, has been incorporated and referenced.     

Lipid peroxidation and antioxidant systems in the liver injury produced by glutathione depleting agents

The mechanisms of the liver damage produced by three glutathione (GSH) depleting agents, bromobenzene, allyl alcohol and diethylmaleate, was investigated. The change in the antioxidant systems represented by alpha-tocopherol (vitamin E) and ascorbic acid were studied under conditions of severe GSH depletion. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. The hepatic level of vitamin E was decreased whenever extensive lipid peroxidation developed. In the case of bromobenzene intoxication, vitamin E decreased before the onset of lipid peroxidation. Changes in levels of the ascorbic and dehydroascorbic acid indicated a redox cycling of vitamin C with the oxidative stress induced by all the three agents. Such a change of the redox state of vitamin C (increase of the oxidized over the reduced form) may be an index of oxidative stress preceding lipid peroxidation in the case of bromobenzene. In the other cases, such a change is likely to be a consequence of lipid peroxidation. Experiments carried out with vitamin E deficient or supplemented diets indicated that the pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. Animals fed a vitamin E supplemented diet had an hepatic vitamin E level double that obtained with a commercial pellet diet. In such animals, bromobenzene and allyl alcohol had only limited toxicity and diethylmaleate none in spite of comparable hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of the toxicity by GSH depleting agents.     

Protective role of vitamin A in the male reproductive toxicity of Hexachlorocyclohexane (HCH) in the rat

The reproductive toxicity of the organochlorine insecticide, hexachlorocyclohexane (HCH), was investigated in male albino rats fed diet free of vitamin A or containing vitamin A at 2000 or 100,000 IU/kg diet. Diets containing 1000 ppm HCH for 7 weeks did not cause testicular toxicity in the vitamin-A-defecient and supplemented rats. However, reproductive toxicity was clearly manifested 2 weeks after withdrawing HCH from the diets and was more pronounced in the vitamin A deficient rats compared to their vitamin A supplemented counterparts. Reduction in the testicular weights was accompanied by atrophy of epididymides and seminal vesicles in the vitamin A deficient rats alone. Inhibition of spermatogenesis was further confirmed by decreased sperm count in the epididymis. Biochemically, the activities of the steroidogenic enzymes were drastically reduced. Supplementation of vitamin A after withdrawal of HCH accelerated the recovery and] restored spermatogenesis and enzyme activities in the deficient rats. These results demonstrate the greater susceptibility of the male reproductive system to HCH toxicity during vitamin A deficiency and also the protective effect of vitamin A supplementation.     

Discovery, modeling, and human pharmacokinetics of N-(2-acetyl-4,6-dimethylphenyl)-3-(3,4-dimethylisoxazol-5-ylsulfamoyl)thiophene-2-carboxamide (TBC3711), a second generation, ETA selective, and orally bioavailable endothelin antagonist

Sitaxsentan (1) (Wu et al. J. Med. Chem. 1997, 40, 1690) is our first endothelin antagonist being evaluated in clinical trials. It has demonstrated biological effects in an acute hemodynamic study in CHF (Givertz et al. Circulation 2000, 101, 2922), an open-label 20-patient pulmonary hypertension trial (Barst et al. Chest 2002, 121, 1860-1868), and a 31-patient trial in essential hypertension (Calhoun et al. AHA Scientific Sessions 2000). In a phase 2b/3 pulmonary arterial hypertension trial, once a day treatment of 100 mg of sitaxsentan statistically significantly improved 6-min walk distance and NYHA class at 12 weeks (Barst et al. Am. J. Respir. Crit. Care Med. 2004, 169, 441). We have since reported on our efforts in generating follow-up compounds (Wu et al. J. Med. Chem. 1999, 42, 4485) and recently communicated that an ortho acyl group on the anilino ring enhanced oral absorption in this category of compounds (Wu et al. J. Med. Chem. 2001, 44, 1211). Here we report an expansion of this work by substituting a variety of electron-withdrawing groups at the ortho position and evaluating their effects on oral bioavailability as well as structure-activity relationships. As a result, TBC3711 (7z) was identified as our second endothelin antagonist to enter the clinic due to its good oral bioavailability (approximately 100%) in rats, high potency (ET(A) IC(50) = 0.08 nM), and optimal ET(A)/ET(B) selectivity (441 000-fold). Compound 7z has completed phase-I clinical development and was well tolerated with desirable pharmacokinetics in humans (t(1/2) = 6-7 h, oral availability > 80%).     

High levels of apoptosis induced by total body irradiation in mice fed a low protein-low vitamin E diet.

Apoptosis in response to total body irradiation (TBI) in mice fed four kinds of diets was assessed. Male mice were fed a low protein-low vitamin E diet, a low protein-basal vitamin E diet, a basal protein-low vitamin E diet or a basal protein-basal vitamin E diet for 2 weeks, then received TBI at a dose of 0, 1 or 4 Gy, and were sacrificed 4 h after TBI. Apoptosis was assessed according to the appearance of DNA fragmentation patterns [DNA laddering and positive in situ end-labeling by terminal transferase (TUNEL) assay] and apoptotic index (AI). Electrophoresis of DNA from the spleen in the groups that did not receive TBI showed no cleavage, whereas that of DNA in the groups with 4 Gy of TBI displayed a ladder pattern, indicative of internucleosomal cleavage characteristic of apoptosis. In the groups with 4 Gy of TBI, apoptosis was enhanced in the low protein-low vitamin E group.     

Comparison of experimentally determined and mathematically predicted percutaneous penetration rates of chemicals

The aim of the study was to evaluate the predictive potential of three different mathematical models for the percutaneous penetration of industrial solvents with respect to our experimental data. Percutaneous penetration rates (fluxes) from diffusion cell experiments of 11 chemicals were compared with fluxes predicted by mathematical models. The chemicals considered were three glycol ethers (2-butoxyethanol, diethylene glycol monobutyl ether and 1-ethoxy-2-propanol), three alcohols (ethanol, isopropanol and methanol), two glycols (ethylene glycol and 1,2-propanediol), one aromatic hydrocarbon (toluene) and two aromatic amines (aniline and o-toluidine). For the mathematical prediction of fluxes, models described by Fiserova-Bergerova et al. (Am J Ind Med 17:617–635 1990), Guy and Potts (Am J Ind Med 23:711–719 1993) and Wilschut et al. (Chemosphere 30:1275–1296 1995) were used. The molecular weights, octanol–water partition coefficients (LogP) and water solubilities of the compounds were obtained from a database for modelling. The fit between the mathematically predicted and experimentally determined fluxes was poor (R ² = 0.04–0.29; linear regression). The flux differences ranged up to a factor of 412. For 4 compounds, the Guy and Potts model showed a closer fit with the experimental flux than the other models. The Wilschut et al. model showed a lower flux difference for 4 compounds as compared to experimental data than the models of Fiserova-Bergerova et al. and Guy and Potts. The Fiserova-Bergerova et al. model showed for 3 compounds a lower flux difference to experimental data than the other models. This study demonstrates large differences between mathematically predicted and experimentally determined fluxes. The percutaneous penetration as determined in diffusion cell experiments may be considerably overestimated as well as underestimated by mathematical models. Although the number of compounds in our comparison study is small, the results point out that none of the mathematical model has significant advantages.     

Effectiveness of mycotoxin sequestration activity of micronized wheat fibres on distribution of ochratoxin A in plasma,liver and kidney of piglets fed a naturally contaminated diet

A study was carried out to determine the ability of dietary micronized wheat fibres (MWF) to decrease the levels of ochratoxin A (OTA) in plasma, kidney and liver of piglets fed a naturally contaminated diet. A total of 96 piglets (weighting 11.4 ± 1.5 kg) were fed one of four different diets for 28 days. Diets included (1) control diet, (2) control diet with MWF (1%), (3) OTA naturally contaminated diet (117.45 ± 4.74 ng/g), (4) OTA naturally contaminated diet (118.13 ± 2.85 ng/g) with MWF (1%). No difference in feed efficiency (P > 0.05) could be observed between the different diets. The absolute weight of kidneys and liver were significantly higher in pigs fed the OTA-contaminated diet (diet 3) as compared to the control diet (diet 1) or to the control diet amended with MWF (diet 2) (P < 0.05). However the use of MWF (diet 4) significantly protected against these weight changes. A significant protective effect of MWF was also observed in terms of OTA concentration in plasma (45.6% decrease), kidney (40.8% decrease) and liver (26.5% decrease). These results suggest that the addition of MWF is effective in decreasing the bioavailability of OTA from contaminated diets in piglets.     

The effects of intermittent nitrogen dioxide exposure on vitamin E-deficient and -sufficient rats

In two separate experiments rats fed vitamin E-deficient, normal or high vitamin E-supplemented diets were intermittently exposed to 15 ppm ± 1.0 ppm nitrogen dioxide (NO₂) over a 5-week (4 days/week, total of 31.5 h exposure) or an 18-week (5 days/week, total of 93.5 h exposure) period. In the 5-week, NO₂-exposed rats, the blood methemoglobin levels were not influenced by NO₂ exposure or the level of vitamin E in the diet. Tissues of the rats exposed to NO₂ for 18 weeks showed some histological changes; in the lung, increased atelectasis and alveolar thickening and in the liver, increased granular changes, karyolysis and karyorhexis. These differences were suppressed by increasing levels of dietary vitamin E. Tissue lipofuscin pigment (LFP) concentrations were not affected by NO₂ exposure or dietary vitamin E. Fatty acid distribution of lung lipid extracts showed no changes due to NO₂ exposure; however, some effects of dietary vitamin E could be seen. The results suggest that intermittent NO₂ exposure, under the described conditions, did not cause ultimate changes of the biochemical parameters measured.