Acute intravenous exposure to silver nanoparticles during pregnancy induces particle size and vehicle dependent changes in vascular tissue contractility in Sprague Dawley rats.

The use of silver nanoparticles (AgNP) raises safety concerns during susceptible life stages such as pregnancy. We hypothesized that acute intravenous exposure to AgNP during late stages of pregnancy will increase vascular tissue contractility, potentially contributing to alterations in fetal growth. Sprague Dawley rats were exposed to a single dose of PVP or Citrate stabilized 20 or 110nm AgNP (700µg/kg). Differential vascular responses and EC50 values were observed in myographic studies in uterine, mesenteric arteries and thoracic aortic segments, 24h post-exposure. Reciprocal responses were observed in aortic and uterine vessels following PVP stabilized AgNP with an increased force of contraction in uterine artery and increased relaxation responses in aorta. Citrate stabilized AgNP exposure increased contractile force in both uterine and aortic vessels. Intravenous AgNP exposure during pregnancy displayed particle size and vehicle dependent moderate changes in vascular tissue contractility, potentially influencing fetal blood supply.

Attenuation of cocaine self-administration by chronic oral phendimetrazine in rhesus monkeys.

Chronic treatment with the monoamine releaser d-amphetamine has been consistently shown to decrease cocaine self-administration in laboratory studies and clinical trials. However, the abuse potential of d-amphetamine is an obstacle to widespread clinical use. Approaches are needed that exploit the efficacy of the agonist approach but avoid the abuse potential associated with dopamine releasers. The present study assessed the effectiveness of chronic oral administration of phendimetrazine (PDM), a pro-drug for the monoamine releaser phenmetrazine (PM), to decrease cocaine self-administration in four rhesus monkeys. Each day, monkeys pressed a lever to receive food pellets under a 50-response fixed-ratio (FR) schedule of reinforcement and self-administered cocaine (0.003-0.56 mg/kg per injection, i.v.) under a progressive-ratio (PR) schedule in the evening. After completing a cocaine self-administration dose-response curve, sessions were suspended and PDM was administered (1.0-9.0 mg/kg, p.o., b.i.d.). Cocaine self-administration was assessed using the PR schedule once every 7 days while food-maintained responding was studied daily. When a persistent decrease in self-administration was observed, the cocaine dose-effect curve was re-determined. Daily PDM treatment decreased cocaine self-administration by 30-90% across monkeys for at least 4 weeks. In two monkeys, effects were completely selective for cocaine. Tolerance developed to initial decreases in food-maintained responding in the third monkey and in the fourth subject, fluctuations were observed that were lower in magnitude than effects on cocaine self-administration. Cocaine dose-effect curves were shifted down and/or rightward in three monkeys. These data provide further support for the use of agonist medications for cocaine abuse, and indicate that the promising effects of d-amphetamine extend to a more clinically viable pharmacotherapy.

Cardiac Ischemia Reperfusion Injury Following Instillation of 20 nm Citrate-capped Nanosilver.

BACKGROUND: Silver nanoparticles (AgNP) have garnered much interest due to their antimicrobial properties, becoming one of the most utilized nano-scale materials. However, any potential evocable cardiovascular injury associated with exposure has not been reported to date. We have previously demonstrated expansion of myocardial infarction after intratracheal (IT) instillation of carbon-based nanomaterials. We hypothesized pulmonary exposure to Ag core AgNP induces a measureable increase in circulating cytokines, expansion of cardiac ischemia-reperfusion (I/R) injury and is associated with depressed coronary constrictor and relaxation responses. Secondarily, we addressed the potential contribution of silver ion release on AgNP toxicity. METHODS: Male Sprague-Dawley rats were exposed to 200 µl of 1 mg/ml of 20 nm citrate-capped Ag core AgNP, 0.01, 0.1, 1 mg/ml Silver Acetate (AgAc), or a citrate vehicle by intratracheal (IT) instillation. One and 7 days following IT instillation the lungs were evaluated for inflammation and the presence of silver; serum was analyzed for concentrations of selected cytokines; cardiac I/R injury and coronary artery reactivity were assessed. RESULTS: AgNP instillation resulted in modest pulmonary inflammation with detection of silver in lung tissue and alveolar macrophages, elevation of serum cytokines: G-CSF, MIP-1α, IL-1ß, IL-2, IL-6, IL-13, IL-10, IL-18, IL-17α, TNFα, and RANTES, expansion of I/R injury and depression of the coronary vessel reactivity at 1 day post IT compared to vehicle treated rats. Silver within lung tissue was persistent at 7 days post IT instillation and was associated with an elevation in cytokines: IL-2, IL-13, and TNFα and expansion of I/R injury. AgAc resulted in a concentration dependent infarct expansion and depressed vascular reactivity without marked pulmonary inflammation or serum cytokine response. CONCLUSIONS: Based on these data, IT instillation of AgNP increases circulating levels of several key cytokines, which may contribute to persistent expansion of I/R injury possibly through an impaired vascular responsiveness.

Vascular Tissue Contractility Changes Following Late Gestational Exposure to Multi-Walled Carbon Nanotubes or their Dispersing Vehicle in Sprague Dawley Rats.

Multi-walled carbon nanotubes (MWCNTs) are increasingly used in industry and in nanomedicine raising safety concerns, especially during unique life-stages such as pregnancy. We hypothesized that MWCNT exposure during pregnancy will increase vascular tissue contractile responses by increasing Rho kinase signaling. Pregnant (17-19 gestational days) and non-pregnant Sprague Dawley rats were exposed to 100 µg/kg of MWCNTs by intratracheal instillation or intravenous administration. Vasoactive responses of uterine, mesenteric, aortic and umbilical vessels were studied 24 hours post-exposure by wire myography. The contractile responses of the vessel segments were different between the pregnant and non-pregnant rats, following MWCNT exposure. Maximum stress generation in the uterine artery segments from the pregnant rats following pulmonary MWCNT exposure was increased in response to angiotensin II by 4.9 mN/mm2 (+118%), as compared to the naïve response and by 2.6 mN/mm2 (+40.7%) as compared to the vehicle exposed group. Following MWCNT exposure, serotonin induced approximately 4 mN/mm2 increase in stress generation of the mesenteric artery from both pregnant and non-pregnant rats as compared to the vehicle response. A significant contribution of the dispersion medium was identified as inducing changes in the contractile properties following both pulmonary and intravenous exposure to MWCNTs. Wire myographic studies in the presence of a Rho kinase inhibitor and RhoA and Rho kinase mRNA/protein expression of rat aortic endothelial cells were unaltered following exposure to MWCNTs, suggesting absent/minimal contribution of Rho kinase to the enhanced contractile responses following MWCNT exposure. The reactivity of the umbilical vein was not changed; however, mean fetal weight gain was reduced with dispersion media and MWCNT exposure by both routes. These results suggest a susceptibility of the vasculature during gestation to MWCNT and their dispersion media-induced vasoconstriction, predisposing reduced fetal growth during pregnancy.

Human exposure and internal dose assessments of acrylamide in food.

This review provides a framework contributing to the risk assessment of acrylamide in food. It is based on the outcome of the ILSI Europe FOSIE process, a risk assessment framework for chemicals in foods and adds to the overall framework by focusing especially on exposure assessment and internal dose assessment of acrylamide in food. Since the finding that acrylamide is formed in food during heat processing and preparation of food, much effort has been (and still is being) put into understanding its mechanism of formation, on developing analytical methods and determination of levels in food, and on evaluation of its toxicity and potential toxicity and potential human health consequences. Although several exposure estimations have been proposed, a systematic review of key information relevant to exposure assessment is currently lacking. The European and North American branches of the International Life Sciences Institute, ILSI, discussed critical aspects of exposure assessment, parameters influencing the outcome of exposure assessment and summarised data relevant to the acrylamide exposure assessment to aid the risk characterisation process. This paper reviews the data on acrylamide levels in food including its formation and analytical methods, the determination of human consumption patterns, dietary intake of the general population, estimation of maximum intake levels and identification of groups of potentially high intakes. Possible options and consequences of mitigation efforts to reduce exposure are discussed. Furthermore the association of intake levels with biomarkers of exposure and internal dose, considering aspects of bioavailability, is reviewed, and a physiologically-based toxicokinetic (PBTK) model is described that provides a good description of the kinetics of acrylamide in the rat. Each of the sections concludes with a summary of remaining gaps and uncertainties.

A physiologically based pharmacokinetic model for ethylene oxide in mouse, rat, and human.

Ethylene oxide (EO) is widely used as a gaseous sterilant and industrial intermediate and is a direct-acting mutagen and carcinogen. The objective of these studies was to develop physiologically based pharmacokinetic (PB-PK) models for EO to describe the exposure-tissue dose relationship in rodents and humans. We previously reported results describing in vitro and in vivo kinetics of EO metabolism in male and female F344 rats and B6C3F1 mice. These studies were extended by determining the kinetics of EO metabolism in human liver cytosol and microsomes. The results indicate enzymatically catalyzed GSH conjugation via cytosolic glutathione S-transferase (cGST) and hydrolysis via microsomal epoxide hydrolase (mEH) occur in both rodents and humans. The in vitro kinetic constants were scaled to account for cytosolic (cGST) and microsomal (mEH) protein content and incorporated into PB-PK descriptions for mouse, rat, and human. Flow-limited models adequately predicted blood and tissue EO levels, disposition, and elimination kinetics determined experimentally in rats and mice, with the exception of testis concentrations, which were overestimated. Incorporation of a diffusion-limited description for testis improved the ability of the model to describe testis concentrations. The model accounted for nonlinear increases in blood and tissue concentrations that occur in mice on exposure to EO concentrations greater than 200 ppm. Species differences are predicted in the metabolism and exposure-dose relationship, with a nonlinear relationship observed in the mouse as a result of GSH depletion. These models represent an essential step in developing a mechanistically based EO exposure-dose-response description for estimating human risk from exposure to EO.

Metabolism and disposition of bisphenol A in female rats.

Bisphenol A (BPA), which is used in the manufacture of polycarbonates, elicits weak estrogenic activity in in vitro and in vivo test systems. The objectives of this study were to compare the patterns of disposition of radioactivity in adult female F-344 and CD rats after oral administration of (14)C BPA (100 mg/kg), to isolate the glucuronide of BPA and to assess its estrogenic activity in vitro, and to evaluate the transfer of radioactivity to pups from lactating dams administered (14)C BPA. Over 6 days, F-344 rats excreted more radioactivity in urine than CD rats. The major metabolite in urine was identified as bisphenol A glucuronide (BPA gluc) by incubation with beta-glucuronidase and (1)H and (13)C NMR spectroscopy. In lactating CD rats administered (14)C BPA (100 mg/kg) by gavage, only a small fraction of the label was found in milk, with 0.95 +/- 0.66, 0.63 +/- 0.13, and 0.26 +/- 0.10 microg equiv/ml (mean +/- SD) from dams collected 1, 8, and 26 h after dosing, respectively. Radioactivity in pup carcasses indicated exposure in the range of microgram equivalents per kilogram; those values ranged from 44.3 +/- 24.4 for pups separated from their lactating dams at 2 h to 78.4 +/- 10.9 at 24 h. BPA gluc was the prominent metabolite in milk and plasma. In test systems for activation of in vitro estrogen receptors alpha and beta, BPA gluc did not show appreciable efficacy at concentrations up to 0.03 mM, indicating that metabolism via glucuronidation is a detoxication reaction.

Hemoglobin adducts from acrylonitrile and ethylene oxide in cigarette smokers: effects of glutathione S-transferase T1-null and M1-null genotypes.

Acrylonitrile (ACN) is used to manufacture plastics and fibers. It is carcinogenic in rats and is found in cigarette smoke. Ethylene oxide (EO) is a metabolite of ethylene, also found in cigarette smoke, and is carcinogenic in rodents. Both ACN and EO undergo conjugation with glutathione. The objectives of this study were to examine the relationship between cigarette smoking and hemoglobin adducts derived from ACN and EO and to investigate whether null genotypes for glutathione transferase (GSTM1 and GSTT1) alter the internal dose of these agents. The hemoglobin adducts N-(2-cyanoethyl)valine (CEVal), which is formed from ACN, and N-(2-hydroxyethyl)valine (HEVal), which is formed from EO, and GST genotypes were determined in blood samples obtained from 16 nonsmokers and 32 smokers (one to two packs/day). Smoking information was obtained by questionnaire, and plasma cotinine levels were determined by immunoassay. Glutathione transferase null genotypes (GSTM1 and GSTT1) were determined by PCR. Both CEVal and HEVal levels increased with increased cigarette smoking dose (both self-reported and cotinine-based). CEVal and HEVal levels were also correlated. GSTM1 and GSTT1 genotypes had little effect on CEVal concentrations. GSTM1 null genotypes had no significant impact on HEVal. However, HEVal levels were significantly elevated in GSTT1-null individuals when normalized to smoking status or cotinine levels. The ratio of HEVal:CEVal was also elevated in GSTT1-null smokers (1.50 +/- 0.57 versus 0.88 +/- 0.24; P = 0.0002). The lack of a functional GSTT1 is estimated to increase the internal dose of EO derived from cigarette smoke by 50-70%.

Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice.

Acrylonitrile (AN) and acrylamide (AM) are commonly used in the synthesis of plastics and polymers. In rodents, AM and AN are metabolized to the epoxides glycidamide and cyanoethylene oxide, respectively. The aim of this study was to determine the role of cytochrome P450 in the metabolism of AM and AN in vivo. Wild-type (WT) mice, WT mice pretreated with aminobenzotriazole (ABT, 50 mg/kg ip, 2 h pre-exposure), and mice devoid of cytochrome P450 2E1 (P450 2E1-null) were treated with 50 mg/kg [(13)C]AM po. WT mice and P450 2E1-null mice were treated with 2.5 or 10 mg/kg [(13)C]AN po. Urine was collected for 24 h, and metabolites were characterized using (13)C NMR. WT mice excreted metabolites derived from the epoxides and from direct GSH conjugation with AM or AN. Only metabolites derived from direct GSH conjugation with AM or AN were observed in the urine from ABT-pretreated WT mice and P450 2E1-null mice. On the basis of evaluation of urinary metabolites at these doses, these data suggest that P450 2E1 is possibly the only cytochrome P450 enzyme involved in the metabolism of AM and AN in mice, that inhibiting total P450 activity does not result in new pathways of non-P450 metabolism of AM, and that mice devoid of P450 2E1 do not excrete metabolites of AM or AN that would be produced by oxidation by other cytochrome P450s. P450 2E1-null mice may be an appropriate model for the investigation of the role of oxidative metabolism in the toxicity or carcinogenicity of these compounds.

Ethylene oxide dosimetry in the mouse.

Ethylene oxide (EO) is a direct-acting mutagen and animal carcinogen used as an industrial intermediate and sterilant with a high potential for human exposure. Understanding the exposure-dose relationship for EO in rodents is critical for developing human EO exposure-dose models. The study reported here examined the dosimetry of EO in male B6C3F1 mice by direct determination of blood EO concentrations. Steady-state blood EO concentrations were measured during a single 4-h nose-only inhalation exposure (0, 50, 100, 200, 300, or 400 ppm EO). In addition, glutathione (GSH) concentrations were measured in liver, lung, kidney, and testis to assess the role of the GSH depletion in the saturable metabolism previously observed in mice (Brown et al., Toxicol. Appl. Pharmacol. 136, 8-19, 1996). Blood EO concentrations were found to increase linearly with exposure concentration up to 200 ppm. Markedly sublinear blood dosimetry was observed at exposure concentrations exceeding 200 ppm. An EO exposure concentration-dependent reduction in tissue GSH levels was observed, with both liver and lung GSH levels significantly depressed at EO exposure concentrations of 100 ppm or greater. Our results also indicate that depletion of GSH is likely responsible for nonlinear dosimetry of EO in mice and that GSH depletion corresponds with reports of dose-rate effects in mice exposed to EO.

Evaluation of the metabolism and hepatotoxicity of styrene in F344 rats, B6C3F1 mice, and CD-1 mice following single and repeated inhalation exposures.

Styrene is used for the manufacture of plastics and polymers. The metabolism and hepatotoxicity (mice only) of styrene was compared in male B6C3F1 mice, CD-1 mice, and F344 rats to evaluate biochemical mechanisms of toxicity. Rats and mice were exposed to 250 ppm styrene for 6 h/day for 1 to 5 days, and liver (mice only) and blood were collected following each day of exposure. Mortality and increased serum alanine aminotransferase (ALT) activity were observed in mice but not in rats. Hepatotoxicity in B6C3F1 mice was characterized by severe centrilobular congestion after one exposure followed by acute centrilobular necrosis. Hepatotoxicity was delayed by 1 day in CD-1 mice, and the increase in ALT and degree of necrosis was less than observed for B6C3F1 mice. Following exposure to unlabeled styrene for 0, 2, or 4 days, rats and mice were exposed to [7-14C]-styrene (60 microCi/mmol) for 6 h. Urine, feces, and expired air were collected for up to 48 h. Most styrene-derived radioactivity was excreted in urine. The time-course of urinary excretion indicates that rats and CD-1 mice eliminated radioactivity at a faster rate than B6C3F1 mice following a single 250 ppm exposure, consistent with a greater extent of liver injury for B6C3F1 mice. The elimination rate following 3 or 5 days of exposure was similar for rats and both mouse strains. Following three exposures, the total radioactivity eliminated in excreta was elevated over that measured for one exposure for both mouse strains. An increased excretion of metabolites on multiple exposure is consistent with the absence of ongoing acute necrosis following 4 to 5 daily exposures. These data indicate that an induction in styrene metabolism occurs after multiple exposures, resulting in an increased uptake and/or clearance for styrene.

Urinary metabolites from F344 rats and B6C3F1 mice coadministered acrylamide and acrylonitrile for 1 or 5 days.

The purpose of this study was to examine the feasibility of using 13C NMR spectroscopy to analyze urinary metabolites produced following coadministration of two structurally similar carbon-13-labeled compounds to rodents. Acrylonitrile (AN) and acrylamide (AM) are used in the chemical industry to manufacture plastics and polymers. These compounds are known to produce carcinogenic, reproductive, or neurotoxic effects in laboratory animals. The potential for human exposure to AN and AM occurs in manufacturing facilities and environmentally. Male F344 rats and B6C3F1 mice were coadministered po [1,2,3-13C]AN (16-17 mg/kg) and [1,2,3-13C]AM (21-22 mg/kg) after 0 or 4 days of administration of unlabeled AN or AM. Urine was collected for 24 h following administration of the 13C-labeled compounds and analyzed by 13C NMR spectroscopy. Rats and mice excreted metabolites derived from glutathione (GSH) conjugation with AM or AN or derived from GSH conjugation with the epoxides cyanoethylene oxide (CEO) or glycidamide (GA). GA and its hydrolysis product were also detected in the urine of rats and mice. For mice, an increased urinary excretion of total AN- and total AM-derived metabolites (p < 0.05) on repeated coadministration suggested a possible increase in metabolism via oxidation. In addition, mice had an increased (p < 0.05) percentage of dose excreted as metabolites derived from GSH conjugation with AM, AN, CEO, or GA after five exposures as compared with one exposure that may be related to a significant increase in the synthesis of GSH or an increase in glutathione transferase activity. The only significant (p < 0.05) increase between one and five exposures for the rat was in the percentage of metabolites produced following conversion of AM to GA. The use of 13C NMR spectroscopy has provided a powerful methodology for elucidation of the metabolism of two 13C-labeled chemicals administered simultaneously.

Characterization of urinary metabolites from Sprague-Dawley rats and B6C3F1 mice exposed to [1,2,3,4-13C]butadiene.

1,3-Butadiene (BD) is used in the production of synthetic rubber and other resins. Carcinogenic effects have been observed in laboratory animals exposed to BD, with mice being more sensitive than rats. Metabolic oxidation of butadiene to epoxides is believed to be a crucial step in the initiation of tumors by BD. However, limited information is available that describes the in vivo metabolism of BD. Male Sprague-Dawley rats and B6C3F1 mice were exposed to 800 ppm [1,2 3,4-13C]butadiene for 5 h, and urine was collected during and for 20 h following exposure. Urinary metabolites were characterized using 1- and 2-dimensional methods of NMR spectroscopy. Three metabolites previously detected in vivo, N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine, N-acetyl-S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine, and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine, were present in both rat and mouse urine, accounting for 87% and 73% of the total metabolites excreted, respectively. A fourth metabolite, previously detected in vitro, 3-butene-1,2-diol, was also present in both rat and mouse urine and comprised 5% and 3% of the total metabolites excreted, respectively. An additional metabolite detected only in mouse urine that is derived from glutathione conjugation with epoxybutene was identified as S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine (4%). N-Acetyl-S-(1-hydroxy-3-butenyl)-L-cysteine (4%), detected in mouse urine, is a thiohemiacetal product of 3-butenal. Additionally, mice excreted N-acetyl-S-(3-hydroxypropyl)-L-cysteine (5%) and N-acetyl-S-(2-carboxyethyl)-L-cysteine (5%), which could be derived from further metabolism of N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine or from glutathione conjugation with acrolein. Mice excreted N-acetyl-S-(1-(hydroxymethyl)-3,4-dihydroxypropyl)-L-cysteine (5%), which could be derived from glutathione conjugation with diepoxybutane (BDE), while rats excreted 1,3-dihydroxypropanone (5%), which may be derived from hydrolysis of BDE. These studies indicate that reactive aldehydes are produced as metabolites of BD in vivo, in addition to the reactive monoepoxide and diepoxide of BD. The greater toxicity of BD in mice compared with rats may be attributed to the greater ability of rats to detoxify BDE via hydrolysis, and/or to the production of reactive aldehydes.

In vivo and in vitro kinetics of ethylene oxide metabolism in rats and mice.

Ethylene oxide (EO) is a direct-acting mutagen and animal carcinogen used as an industrial intermediate and sterilant with a high potential for human exposure. Kinetics of EO metabolism in rodents can be used to develop human EO dosimetry models. This study examined the kinetics of EO metabolism in vivo and in vitro in male and female F-344 rats and B6C3F1 mice. In vivo studies measured blood and tissue EO levels during and 2-20 min following whole-body inhalation exposure (4 hr, 100 or 330 ppm EO). At 100 ppm EO, the half-life of elimination (t1/2) in rats was 13.8 +/- 0.3 (mean +/- SD) and 10.8 +/- 2.4 min for males and females, respectively, compared to a t1/2 in mice of 3.12 +/- 0.2 and 2.4 +/- 0.2 min in males and females, respectively. On exposure to 330 ppm EO, the t1/2 in mice increased approx twofold, while no change in t1/2 was observed in rats. In vitro kinetic parameters (Vmax and KM) of EO metabolism were determined using tissue cytosol and microsomes. EO metabolism in vitro occurred primarily via cytosolic glutathione S-transferase-mediated EO-GSH conjugation (cGST-EO), with highest activity in the liver. Liver cGST-EO activity (Vmax) was 258 +/- 86.9 and 287 +/- 49.0 nmol/mg protein/min (mean +/- SD) in male and female mice, respectively, compared to 52.7 +/- 10.8 and 29.3 +/- 4.9 in male and female rats, respectively. In rats, but not mice, there was a statistically significant (p < 0.05) gender difference in the Vmax for liver cGST. The KM for liver cGST-EO was approximately 10 mM in both species. The higher Vmax values observed in mice are consistent with the more rapid elimination of EO observed for this species in vivo compared to rats.

A physiologically based dosimetry description of acrylonitrile and cyanoethylene oxide in the rat.

The cytochrome P450-mediated oxidation of acrylonitrile (ACN) to the mutagen 2-cyanoethylene oxide (CEO) is thought to be important for the carcinogenic effects of ACN in rats, while glutathione (GSH) conjugation of ACN and CEO is regarded as detoxication. A physiologically based dosimetry description for ACN and CEO in the male F-344 rat has been developed from in vitro data and studies of the iv pharmacokinetics of ACN and CEO. The dosimetry description includes tissue partition coefficients and in vitro estimates of the rates of reaction of ACN and CEO with hemoglobin and blood macromolecules and the reaction of CEO with tissue GSH. Metabolic parameters for ACN and CEO were estimated from iv pharmacokinetic studies. Rats were given bolus doses of 3.4, 47, 55, or 84 mg ACN/kg via the femoral vein and blood samples were collected at selected time points. ACN and CEO blood concentrations were determined by gas chromatography. The iv pharmacokinetics of CEO were also determined using 0.6 or 5.3 mg CEO/kg. ACN elimination from blood was described by saturable P450 epoxidation (Vmax of 6.5 mg/hr/kg and Km of 1.5 mg/liter) and first-order GSH conjugation (30 hr-1/kg). CEO elimination was described by first-order GSH conjugation (750 hr-1/kg). Calculation of hepatic clearance values shows first-pass hepatic extractions of 61 and 90% for ACN and CEO, respectively. The dosimetry description accurately simulated the dose-dependent urinary excretion of ACN metabolites derived from epoxidation to CEO and from direct GSH conjugation of ACN. The dose-dependent formation of hemoglobin adducts from ACN was also well simulated.

Dose effects on the excretion of urinary metabolites of 2-[1,2,methoxy-13C]methoxyethanol in rats and mice.

The administration of 2-methoxyethanol (2-ME) to pregnant rats, mice, or primates results in developmental toxicity. To assess the role of metabolism in the adverse response of 2-ME, carbon-13 NMR spectroscopy was used to examine, directly in the urine, metabolites produced after administering high (250 mg/kg) and low (25 mg/kg) doses of 2-[1,2,methoxy-13C]ME to pregnant CD-1 mice and male Fischer 344 rats. The high dose elicits teratogenic effects in mice and testicular toxicity in rats. The urinary disposition was also examined after dosing pregnant CD-1 mice with a developmentally toxic level of 2-ME together with serine or acetate (known attenuators of 2-ME embryotoxicity). Seven novel metabolites were found in rat urine, consistent with those assigned in our previous studies with mice. Metabolite composition was compared for the different dosing regimens. A lower percentage of metabolites derived after conversion of 2-ME to 2-methoxyacetic acid (2-MAA) was found following concurrent administration of 2-ME with acetate, D-serine, or L-serine. These differences are mainly attributed to higher levels of ethylene glycol and/or glycolic acid that arise for the 2-ME administrations with any of the attenuators. Acetate together with 2-ME also reduced the percentage of metabolites incorporated into intermediary metabolism. These data indicate that attenuators of 2-ME teratogenic effects may alter metabolism and distribution by decreasing the conversion of 2-ME to 2-MAA, decreasing the conversion of 2-MAA to a coenzyme A thioester (2-methoxyacetyl approximately CoA), altering the utilization of the coenzyme A thioester, and/or increasing the conversion of 2-ME to ethylene glycol and its further metabolism. These changes in metabolism may contribute to the attenuating effects of these agents on 2-ME.

Reconsideration of the genetic risk assessment for ethylene oxide exposures.

The US Environmental Protection Agency (EPA) developed a genetic risk assessment model for exposures to ethylene oxide utilizing data on the induction of reciprocal translocations in male germ cells [Rhomberg et al. 1990]. This particular approach served as a reasonable initial attempt, albeit somewhat limited with regard to endpoint and only partially attentive to the mechanisms of induction of genetic alterations and the behavior of chromosomes during meiosis. The present paper discusses the scientific basis for a reassessment of the EPA model, providing data and hypotheses related to effective dose to the target cells and shape of the dose-response relationship at low doses, and dose rates. While the present genetic risk assessment approach is discussed in terms of ethylene oxide, it would be applicable to most mutagenic chemicals. The outcome of the discussion is that the genetic risk for exposed males from reciprocal translocation induction will be negligible at low doses since the dose-response curve is likely to be a function of the square of the dose. In addition, the proportion of genetically unbalanced live born offspring in humans arising from reciprocal translocation carriers is less than 10% of the frequency formed through meiotic segregation and fertilization for such carriers. Simply from a consideration of mechanism--namely, the very high probability of DNA repair prior to the next S-phase for a resting oocyte--it would be predicted that there would be a very low to negligible frequency of translocations in female germ cells from ethylene oxide exposure. It is further stressed that additional components of a genetic risk model require a consideration of all germ cell stages in the male, and the inclusion of calculations for point and deletion mutations. Some indications of likely response are presented with these points in mind.

Monitoring exposure to acrylonitrile using adducts with N-terminal valine in hemoglobin.

Human exposure to acrylonitrile (ACN), a carcinogen in rats, may occur in industrial settings, through waste water and tobacco smoke. ACN is an electrophilic compound and binds covalently to nucleophilic sites in macromolecules. Measurements of adducts with hemoglobin could be utilized for improved exposure assessments. In this study, a method for quantification of N-(2-cyanoethyl)valine (CEVal), the product of reaction of ACN with N-terminal valine in hemoglobin has been developed. The method is based on the N-alkyl Edman procedure, which involves derivatization of the globin with pentafluorophenyl isothiocyanate and gas chromatographic-mass spectrometric analysis of the resulting thiohydantoin. An internal standard was prepared by reacting valylglycylglycine with [2H3]ACN, spiked with [14C]ACN to a known sp. act. Levels of CEVal were measured in globin from rats exposed to 3-300 p.p.m. ACN in drinking water for 105 days and from humans (four smokers and four non-smokers). CEVal was detected at all exposure levels in the drinking water study. The relationship between adduct level and water concentration was linear at concentrations of 10 p.p.m. (corresponding to an average daily uptake of c. 0.74 mg ACN/kg body wt during the 65 days prior to sacrifice) and below, with a slope of 37.7 pmol CEVal/g globin/p.p.m. At higher concentrations, adduct levels increased sublinearly, indicating saturation of a metabolic process for elimination of ACN. Comparison of adduct formation with the estimated dose (mg/kg/day) of ACN indicated that at low dose (0-10 p.p.m.) CEVal = 0.508 x ACN dose + 0.048 and at high dose (35-300 p.p.m.) CEVal = 1.142 x ACN dose - 1.098. Globin from the smokers (10-20 cigarettes/day) contained about 90 pmol CEVal/g, whereas the adduct levels in globin from non-smokers were below the detection limit. The analytical sensitivity should be sufficient to allow monitoring of occupationally exposed workers at levels well below the current Occupational Safety and Health Administration standard of 2 p.p.m.

Development of methods for measuring biological markers of formaldehyde exposure.

Formaldehyde, a widely used industrial chemical that is also present in automobile exhaust, causes nasal tumors in rats and mice after prolonged inhalation exposure to high concentrations. The induction of squamous cell carcinomas in rats by formaldehyde displayed a highly nonlinear dose response with a disproportionately large number of tumors at higher exposure concentrations. A sufficient amount of formaldehyde reaching target cells, and the saturation of formaldehyde metabolism to formate can increase the covalent binding of formaldehyde to DNA. The carcinogenicity of formaldehyde may result from its ability to induce DNA-protein cross-links and/or hydroxymethyl adducts in DNA. Measuring these products can indicate the dose of this carcinogen at a critical target site, and such assessment has been conducted for formaldehyde by measuring DNA-protein cross-links. The objective of this study was to develop methods for measuring hydroxymethyl adducts in DNA that do not require the use of radiolabeled formaldehyde. The detection of N6-hydroxymethyldeoxyadenosine and N2-hydroxymethyldeoxyguanosine, the major adducts formed by the reaction of formaldehyde with DNA in vitro, is complicated by their instability. The stabilization of hydroxymethyl adducts by reaction with sodium bisulfite in aqueous solution at 4 degrees C before isolating DNA from homogenates was investigated. On treatment of calf thymus DNA or isolated rat liver nuclei with [14C]formaldehyde, followed by reaction with bisulfite and isolation of DNA, radioactive peaks corresponding in retention time to N6-sulfomethyldeoxyadenosine and N2-sulfomethyldeoxy-guanosine were detected by high-performance liquid chromatography of nucleoside digests. However, on treatment of cultured lymphoblasts with [14C]formaldehyde, extensive metabolic incorporation of radioactivity into normal nucleosides precluded the detection of the derivatives. Methods for detecting these derivatives that do not involve the use of radiolabeled formaldehyde, such as 32P-postlabeling and electrophore postlabeling, were investigated. For electrophore postlabeling, several reactions for preparing a derivative suitable for analysis by gas chromatography with mass spectrometry were investigated unsuccessfully. For 32P-postlabeling, a method was developed for detecting sulfomethyldeoxyadenosine 3',5'-diphosphate that involved separating sulfomethyldeoxyadenosine 3'-monophosphate from normal nucleotides by reverse-phase high-performance liquid chromatography using two columns with column switching. The purified adduct fractions were subjected to 32P-postlabeling, and the labeled adduct was separated by two-dimensional thin-layer chromatography on polyethyleneimine-cellulose plates. The adduct spots were quantitated by comparing them with standards labeled directly or mixed with normal nucleotide 3'-monophosphates and separated by high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of phosphodiester adducts produced by the reaction of cyanoethylene oxide with nucleotides.

Cyanoethylene oxide (CEO), a putative toxic and carcinogenic metabolite of acrylonitrile, is a direct-acting mutagen. The focus of this study was to elucidate potential adducts responsible for the mutagenic effect of CEO by characterizing products from the reaction of CEO with nucleotides. The reaction of CEO with the 5'-monophosphates of deoxyguanosine, deoxyadenosine, deoxycytidine or deoxythymidine resulted in the formation of at least one adduct for each nucleotide. Using two-dimensional NMR spectroscopy and fast atom bombardment mass spectrometry, CEO-nucleotide adducts (approximately 25% modification) were characterized as 2-cyano-2-hydroxyethyl phosphodiesters. The isolate from the reaction of deoxyguanosine-5'-monophosphate (dGMP) with CEO contained a second adduct, identified as N7-(2-cyano-2-hydroxyethyl)-dGMP. Single and double strand breaks, which were observed in supercoiled pBR322 plasmid DNA exposed to CEO (> 50 mM), may arise following formation of cyanohydroxyethyl phosphotriester adducts. The characterization of these phosphodiester adducts in vitro may provide insight into the intermediates responsible for the genotoxic effect of CEO in vivo.