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Chlorine is a commonly used, reactive compound to which humans can be exposed via accidental or intentional release resulting in acute lung injury. Formulations of rolipram (a phosphodiesterase inhibitor), triptolide (a natural plant product with anti-inflammatory properties), and budesonide (a corticosteroid), either neat or in conjunction with poly(lactic:glycolic acid) (PLGA), were developed for treatment of chlorine-induced acute lung injury by intramuscular injection. Formulations were produced by spray-drying, which generated generally spherical microparticles that were suitable for intramuscular injection. Multiple parameters were varied to produce formulations with a wide range of in vitro release kinetics. Testing of selected formulations in chlorine-exposed mice demonstrated efficacy against key aspects of acute lung injury. The results show the feasibility of developing microencapsulated formulations that could be used to treat chlorine-induced acute lung injury by intramuscular injection, which represents a preferred route of administration in a mass casualty situation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo. Five groups of rats were orally administered a single dose of glycidol ranging from 0 to 75mg/kg bw, and diHOPrVal levels were measured 24h after administration. A dose-dependent increase in diHOPrVal levels was observed with high linearity (R(2)=0.943). Blood sampling at different time points (1, 10, 20, or 40days) from four groups administered glycidol at 12mg/kg bw suggested a linear decrease in diHOPrVal levels compatible with the normal turnover of rat erythrocytes (life span, 61days), with the calculated first-order elimination rate constant (kel) indicating that the diHOPrVal adduct was chemically stable. Then, we measured the second-order rate constant (kval) for the reaction of glycidol with N-terminal valine in rat and human hemoglobin in in vitro experiments with whole blood. The kval was 6.7±1.1 and 5.6±1.3 (pmol/g globin per μMh) in rat and human blood, respectively, indicating no species differences. In vivo doses estimated from kval and diHOPrVal levels were in agreement with the area under the (concentration-time) curve values determined in our earlier toxicokinetic study in rats. Our results indicate that diHOPrVal is a useful biomarker for quantification of glycidol exposure and for risk assessment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxic mechanisms, particularly toxicokinetics and bioavailability. This study compares the toxicokinetics of AA and its epoxide metabolite glycidamide (GA) in serum and tissues of male and female B6C3F1 mice following acute dosing by intravenous, gavage, and dietary routes at 0.1 mg/kg AA or intravenous and gavage dosing with an equimolar amount of GA. AA was rapidly absorbed from oral dosing, was widely distributed to tissues, was efficiently converted to GA, and increased levels of GA-DNA adducts were observed in liver after complete elimination from serum. GA dosing also resulted in rapid absorption, wide distribution to tissues, and produced liver DNA adduct levels that were approximately 40% higher than those from an equimolar dose of AA. While oral administration was found to attenuate AA bioavailability to 23% from the diet and to 32-52% from aqueous gavage, a first-pass effect or other kinetic change resulted in higher relative internal exposure to GA when compared to the intravenous route. A similar effect on relative GA exposure was also evident as the administered dose was reduced, which suggests that as dosing rate decreases, the conversion of AA to GA is more efficient. These findings are critical to the assessment of genotoxicity of AA at low doses in the food supply, which appears to depend on total exposure to GA.

Styrene is used in the manufacture of plastics and polymers and in the boat-building industry. The major metabolic route for styrene in rats, mice, and humans involves conversion to styrene-7,8-oxide (SO). The purpose of this study was to evaluate blood SO, SO-hemoglobin (SO-Hb) adducts, and urinary metabolites in styrene-exposed human volunteers and to compare these results with data previously obtained for rodents. Four healthy male volunteers were exposed for 2 h during light physical exercise to 50 ppm (13)C(8)-styrene vapor via a face mask. Levels and time profiles of styrene in exhaled air, blood, and urine (analyzed by GC) and urinary excretion patterns of mandelic acid and phenylglyoxylic acid in urine (analyzed by HPLC) were comparable to previously published volunteer studies. Maximum levels of SO in blood (measured by GC-MS) of 2.5-12.2 (average 6.7) nM were seen after 2 h, i.e., in the first sample collected after exposure had ended. The styrene blood level in humans was about 1.5 to 2 times higher than in rats and 4 times higher than in mice for equivalent styrene exposures. In contrast the SO levels in human blood was approximately fourfold lower than in mice. The level of hydroxyphenethylvaline (determined by GC-MS-MS) in pooled blood collected after exposure was estimated as 0.3 pmol/g globin corresponding to a SO-Hb adduct increment of about 0.003 pmol/g and ppmh. NMR analyses of urine showed that a major portion (> 95%) of the excreted (13)C-derived metabolites was derived from hydrolysis of SO, while only a small percentage of the excreted metabolites (< 5%) was derived from metabolism via phenylacetaldehyde. Signals consistent with metabolites derived from other pathways of styrene metabolism in rodents (such as glutathione conjugation with SO or ring epoxidation) were not detected., (Copyright 2000 Academic Press.)

Measurement of specific adducts to hemoglobin can be used to establish the dosimetry of electrophilic compounds and metabolites in experimental animals and in humans. The purpose of this study was to investigate the dose response for adduct formation and persistence in rats and mice during long-term low-level exposure to butadiene by inhalation. Adducts of 3,4-epoxy-1-butene, the primary metabolite of butadiene, with N-terminal valine in hemoglobin were determined in male B6C3F1 mice and male Sprague-Dawley rats following exposure to 0, 2, 10, or 100 ppm of 1,3-butadiene, 6 h/day, 5 days/week for 1, 2, 3, or 4 weeks. Blood samples were collected from groups of five mice and three rats at the end of each week during the 4 weeks of exposure and weekly for 3 weeks following the end of the 4-week exposure period. The increase and decrease, respectively, of the adduct levels during and following the end of the 4-week exposure followed closely the theoretical curve for adduct accumulation and removal for rats and mice, thereby demonstrating that the adducts are chemically stable in vivo and that the elimination follows the turnover of the red blood cells. The adduct level increased linearly with butadiene exposure concentration in the mice, whereas a deviation from linearity was observed in the rats. For example, after exposure to 100 ppm butadiene, the epoxybutene-hemoglobin adduct levels were about four times higher in mice than in rats; at lower concentrations of butadiene, the species difference was less pronounced. Blood concentrations of epoxybutene, estimated from hemoglobin adduct levels, were in general agreement with reported concentrations in mice and rats exposed by inhalation to 62.5 ppm. These studies show that adducts of epoxybutene with N-terminal valine in hemoglobin can be used to predict blood concentration of epoxybutene in experimental animals.

Butadiene and styrene are a mixture of hazardous air pollutants found in the workplace of industries producing polymers such as styrene-butadiene rubber. Both butadiene and styrene require metabolic activation to exert their genotoxic effect; therefore metabolic interactions may influence their genotoxicity. Our objective was to quantitate potential metabolic interactions in mice exposed to a mixture of butadiene and styrene. The rate of metabolism of butadiene and styrene was estimated from the steady-state rate of uptake of the chemicals by male B6C3F1 mice exposed for 8 hr in a dynamic, whole-body inhalation system to 100 or 1000 ppm butadiene in combination with 0, 50, 100, or 250 ppm styrene. Styrene, styrene oxide, 1,2-epoxy-3-butene, and 1,2:3,4-diepoxybutane concentrations in blood were measured by gas chromatography-mass spectrometry at 2, 4, 6, and 8 hr of exposure. As the styrene concentration in the mixture increased, the rate of butadiene metabolism was inhibited up to 48%. 1,2-Epoxy-3-butene blood concentrations were increased by approximately 1.5-fold; however, 1,2:3,4-diepoxybutane blood concentrations were unaffected. Styrene uptake in the inhalation system was inhibited slightly by exposure with butadiene, but styrene blood concentrations increased significantly as the butadiene concentration in the mixture increased to 1000 ppm. Blood concentrations of styrene oxide increased approximately 1.6-fold for the 250-ppm styrene exposures when the butadiene concentration was increased from 0 to 1000 ppm. The data suggest that metabolic interactions occurred among the reactive metabolites (e.g., competition for detoxication pathways) as well as between butadiene and styrene in mice exposed to mixtures of butadiene and styrene. However, metabolic interactions were significant only at concentrations of butadiene and styrene higher than those typically observed in the workplace of industries producing polymers of butadiene and styrene.

Inhalation exposure to styrene at concentrations that cause metabolic saturation results in significantly greater hepatotoxicity in B6C3F1 mice than in Swiss mice; females of both strains are more susceptible than males. These studies were conducted to investigate the mouse strain and gender differences in susceptibility to hepatotoxicity caused by repeated exposure to styrene at concentrations that do not cause metabolic saturation. Male and female B6C3F1 and Swiss mice (8 weeks old) were exposed to 0, 150, or 200 ppm styrene for 6 hr/day, 5 days/week, for up to 2 weeks. Changes in body and liver weights, serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) levels, liver histopathology, and total liver glutathione (GSH) were evaluated after 2, 3, 5, and 10 exposures (six mice/sex/strain/time point/concentration). Blood levels of styrene and styrene-7,8-oxide (SO) were measured in mice exposed to 200 ppm styrene for 2,3, or 5 days (six mice/sex/strain/time point/concentration). Serum ALT and SDH levels were significantly elevated only in female B6C3F1 mice after 3 exposures to 200 ppm styrene; enzyme levels had returned to control levels when measured after 5 and 10 exposures. Degeneration and coagulative necrosis of centrilobular hepatocytes were observed in female B6C3F1 mice exposed 2, 3, and 5 days to 150 or 200 ppm styrene; incidences of these lesions were greater in the 200 ppm than in the 150 ppm dose group. After 10 days of exposure to 150 or 200 ppm styrene, hepatocellular lesions had resolved, although a residual chronic inflammation was present in livers of most female B6C3F1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Styrene is a commercially important chemical used in the production of plastics and resins. In initial short-term styrene inhalation studies, toxicity was significantly greater in male B6C3F1 mice than in females, suggesting that males may metabolize styrene more extensively and/or may be less able to detoxify reactive metabolites. In addition, a nonlinear dose-response was observed where toxicity and mortality were greater in mice exposed to 250 ppm than in those exposed to 500 ppm. These studies were conducted to investigate potential mechanism(s) for sex differences and the nonlinear dose-response in styrene toxicity by evaluating the effects of repeated styrene exposure on styrene oxide production, hepatic GSH availability, and hepatotoxicity in male and female B6C3F1 mice. Mice (36/sex/dose) were exposed to 0, 125, 250, or 500 ppm styrene 6 hr/day for up to 3 days. Styrene exposure caused increased mortality and hepatotoxicity (centrilobular necrosis, increased serum liver enzymes) in males and females after one or two exposures to 250 and 500 ppm. Hepatic GSH levels were decreased in a dose-dependent manner in males and females. After one exposure, GSH levels in males rebounded above controls in all dose groups. After three exposures to 125 or 250 ppm males appeared to maintain GSH levels; GSH was still decreased in the 500 ppm group. GSH levels in females were decreased after each exposure in all dose groups to lower levels than in males, and did not rebound above controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhalation toxicity studies were conducted to evaluate mouse strain differences in the susceptibility to styrene vapors. Male and female B6C3F1, C57BL/6, Swiss, and DBA/2 mice (8 weeks old) were exposed to 0, 125, 250, or 500 ppm styrene 6 hr/day, for 4 days (20/sex/dose). Histopathological changes and changes in liver weights were evaluated as a measure of hepatotoxicity. Styrene uptake and styrene-7,8-oxide (SO) formation were estimated by measuring levels of styrene and SO in blood. An estimate of SO detoxification by conjugation with GSH was obtained by measuring hepatic GSH depletion. In general, mortality, increased liver weights, and hepatocellular necrosis were observed in the 250 and 500 ppm dose groups for all strains and both sexes. Considerable sex and strain differences were observed. Mortality, increased liver weights, and hepatocellular necrosis were greatest in B6C3F1 and C57BL/6 mice in the 250 ppm dose group and in males; hepatotoxicity was similar in both strains. Swiss mice exhibited dose-dependent increases in mortality, liver weights, and in hepatocellular necrosis, with only slight sex differences at early time points. Hepatotoxicity in DBA/2, B6C3F1, and C57BL/6 strains was greater at 250 than 500 ppm; however, toxicity was less severe in DBA/2 than in other strains based on absence of mortality in either sex and less extensive liver necrosis at both 250 and 500 ppm. Blood styrene and SO levels did not correlate well with strain differences in toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobin (Hb) adduct determinations were used to monitor occupational exposure to acrylamide (AA) and acrylonitrile (AN). Forty-one workers in a factory in the People's Republic of China who were involved in the synthesis of AA by catalytic hydration of AN and the manufacturing of polyacrylamides were studied. Ten nonexposed workers in the same city served as controls. AA and AN exposures were monitored using the modified Edman degradation procedure for the determination of their respective Hb adducts to N-terminal valine. The adduct levels in the exposed workers were 0.3-34 nmol/g Hb for AA and 0.02-66 nmol/g Hb for AN, as determined by gas chromatography-mass spectrometry (GC-MS). The formation of glycidamide (GA), the epoxide metabolite of AA, in humans was demonstrated by GC-MS analysis of its Hb adduct to N-terminal valine following acid hydrolysis, ion-exchange chromatography, and derivatization. The GA adduct was detected in samples from the exposed persons with levels of 1.6-32 nmol/g Hb. There was a linear relationship between the AA and GA adduct levels (r = 0.96) and the ratio of the in vivo doses of GA and AA was 3:10. These results suggest that AA is metabolized to GA in humans, as had previously been shown in the rat. The high AA adduct levels in the exposed workers, as compared to those expected from air concentrations, indicate that dermal exposure may contribute significantly to the total uptake of AA. The average daily in vivo doses of AA and GA in the highest exposed workers were comparable to the in vivo doses in rats injected with 3 mg/kg AA. Since a regimen of 2 mg/kg/day is known to cause a significant increase of tumors in rats, preventive measures may be necessary for humans exposed to high levels of AA in industrial settings.

A method was developed for the determination of hemoglobin (Hb) adducts formed by the neurotoxic agent acrylamide and its mutagenic epoxide metabolite glycidamide. The method was based on simultaneous measurements of the cysteine adducts formed by these two agents by means of gas chromatography/mass spectrometry in hydrolyzed hemoglobin samples. Rats were injected ip with acrylamide or glycidamide in doses ranging from 0 to 100 mg/kg body wt, and the hemoglobin adduct levels were determined. The hemoglobin binding index of acrylamide to cysteine was found to be 6400 pmol (g Hb)-1/mumol (kg body wt)-1, higher than for any other substance studied so far in the rat, and 1820 pmol (g Hb)-1/mumol (kg body wt)-1 for glycidamide. In rats injected with acrylamide, formation of adducts of the parent compound was approximately linear with dose (0-100 mg/kg), whereas adducts of the epoxide metabolite glycidamide generated a concave curve, presumably reflecting the Michaelis-Menten kinetics of its formation. On the basis of the rate constants for cysteine adduct formation determined in vitro, the first-order rates of elimination of acrylamide and glycidamide from the blood compartment of rats were estimated to be 0.37 and 0.48 hr-1, respectively, using a linear kinetic model. It was further estimated that the percentage of acrylamide converted to glycidamide in the rat decreased from 51% following administration of 5 mg/kg to 13% after a dose of 100 mg/kg. Subchronic treatment of rats with acrylamide (10 mg/kg/day for 10 days or 3.3 mg/kg/day for 30 days) confirmed that the conversion rate of acrylamide to glycidamide, as determined from hemoglobin adduct formation, is higher at low-administered doses. These findings suggest that dose-rate effects may significantly affect risk estimates of this compound and that different low-dose extrapolation procedures should be employed for effects induced by the parent compound acrylamide and those induced by the metabolite glycidamide.