Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Challenges facing sterilization and depyrogenation of nanoparticles: effects on structural stability and biomedical applications.

This review outlines and compares techniques that are currently available for the sterilization of nanoparticles and addresses the topic of endotoxin contamination. Several techniques are available for the removal of microbial contamination from nanoparticles developed for use in nanomedicine applications. These techniques include filtration, autoclaving and irradiation, as well as formaldehyde, ethylene oxide and gas plasma treatments. Of these sterilization methodologies, filtration may potentially remove microbial contamination without altering the physicochemical properties of the carrier nanoparticles, nor affecting their toxicity and functionality. However, no single process may be applied to all nanoparticle preparations and, therefore, it is recommended that each nanoparticle-drug system be validated on a case-by-case basis. From the clinical editor: This comprehensive review covers the currently available methods for removal of microbial contaminations from nanoparticles for nanomedicine applications. The review highlights the pros and cons of each available method. Authors conclude that there is no single best method and recommend a customized approach for each nanoparticle system.

Quantitative cancer risk assessment for ethylene oxide inhalation in occupational settings.

The estimated occupational ethylene oxide (EO) exposure concentrations corresponding to specified extra risks are calculated for lymphoid mortality as the most appropriate endpoint, despite the lack of a statistically significant exposure-response relationship. These estimated concentrations are for occupational exposures--40 years of occupational inhalation exposure to EO from age 20 to age 60 years. The estimated occupational inhalation exposure concentrations (ppm) corresponding to specified extra risks of lymphoid mortality to age 70 years in a population of male and female EO workers are based on Cox proportional hazards models of the most recent updated epidemiology cohort mortality studies of EO workers and a standard life-table calculation. An occupational exposure at an inhalation concentration of 2.77 ppm EO is estimated to result in an extra risk of lymphoid mortality of 4 in 10,000 (0.0004) in the combined worker population of men and women from the two studies. The corresponding estimated concentration decreases slightly to 2.27 ppm when based on only the men in the updated cohorts combined. The difference in these estimates reflects the difference between combining all of the available data or focusing on only the men and excluding the women who did not show an increase in lymphoid mortality with EO inhalation exposure. The results of sensitivity analyses using other mortality endpoints (all lymphohematopoietic tissue cancers, leukemia) support the choice of lymphoid tumor mortality for estimation of extra risk.

Reciprocal translocations in somatic and germ cells of mice chronically exposed by inhalation to ethylene oxide: implications for risk assessment.

Groups of male B6C3F1 mice were exposed by inhalation to 0, 25, 50, 100 or 200 p.p.m. ethylene oxide (EO) for up to 48 weeks (6 hours/day, 5 days/week). Animals were sacrificed at 6, 12, 24 and 48 weeks after the start of the exposure for analyses of reciprocal translocations in peripheral blood lymphocytes and germ cells. The frequency of the total chromosomal aberrations in the peripheral blood lymphocytes was significantly increased at the 100 and 200 p.p.m. exposure concentrations at the 12-week time point, at 50, 100 and 200 p.p.m. at the 24-week time point and at all EO concentrations at the 48-week time point. The frequency of stable reciprocal translocations, which can be used as biomarkers, was increased (P < 0.05) at 100 and 200 p.p.m. at the 12-week time point, at 100 and 200 p.p.m. at the 24-week time point and at 50, 100 and 200 p.p.m. at the 48-week time point. No statistically significant increase could be observed in translocation frequencies at the 6-week time point in the peripheral blood lymphocytes. The exposure-response curves were non-linear when the frequencies of translocations were plotted against EO exposure durations or against EO exposure concentrations. There was no effect of exposure concentration rate on reciprocal translocation frequency. Reciprocal translocations induced in spermatogonial stem cells (observed at the sprematocyte stage) showed significant increases in translocation frequencies over controls at all EO concentrations at 48 weeks. However, increases were small and they did not occur in a dose-responsive manner. The statistically significant increase observed at 12 weeks in the spermatocytes was equivocal. This study provides low-level chronic exposure somatic cytogenetic data generated in mice that can be used to support the shape of the tumour dose-response in rodents and humans The germ cell cytogenetic data are discussed in terms of its relevance for a threshold response for genetic effects at low exposures.

Genotoxicity as a toxicologically relevant endpoint to inform risk assessment: A case study with ethylene oxide.

The purpose of the present investigation is to analyze the in vivo genotoxicity dose-response data of ethylene oxide (EO) and the applicability of the derived point-of-departure (PoD) values when estimating permitted daily exposure (PDE) values. A total of 40 data sets were identified from the literature, and benchmark dose analyses were conducted using PROAST software to identify a PoD value. Studies employing the inhalation route of exposure and assessing gene or chromosomal mutations and chromosomal damage in various tissues were considered the most relevant for assessing risk from EO, since these effects are likely to contribute to adverse health consequences in exposed individuals. The PoD estimates were screened for precision and the values were divided by data-derived adjustment factors. For gene mutations, the lowest PDE was 285 parts per trillion (ppt) based on the induction of lacI mutations in the testes of mice following 48 weeks of exposure to EO. The corresponding lowest PDE value for chromosomal mutations was 1,175 ppt for heritable translocations in mice following 8.5 weeks of EO exposure. The lowest PDE for chromosomal aberrations was 238 ppt in the mouse peripheral blood lymphocytes following 48 weeks of inhalation exposure. The diverse dose-response data for EO-induced genotoxicity enabled the derivation of PoDs for various endpoints, tissues, and species and identified 238 ppt as the lowest PDE in this retrospective analysis.

Synthesis of multimodal polymersomes for targeted drug delivery and MR/fluorescence imaging in metastatic breast cancer model.

The objective of the current study is to design and delivery of targeted PEG-PCL nanopolymersomes encapsulated with Gadolinium based Quantum Dots (QDs) and Doxorubicin (DOX) as magnetic resonance-florescence imaging and anti-cancer agent. Diagnostic and therapeutic efficiency of the prepared theranostic formulation was evaluated in vitro and in vivo. Hydrophobic QDs based on indium-copper-gadolinium-zinc sulfide were synthesized and characterized extensively. Hydrophobic QDs and hydrophilic DOX were loaded in PEG-PCL polymersomes through double emulsion method. Drug release pattern was studied in both citrate (pH 5.4) and phosphate (pH 7.4) buffer during 10 days. Both fluorescence and magnetic properties of bare QDs and prepared formulations were studied entirely. AS1411 DNA aptamer was covalently attached to the surface of polymersomal formulation in order to prepare targeted drug delivery system. Cellular cytotoxicity and cellular uptake analysis were performed in both nucleolin positive (MCF7 and 4T1) and nucleolin negative (CHO) cell lines. After in vitro evaluations, anti-tumor efficiency and diagnostic capability of the formulation was investigated in 4T1 tumor baring mice. Scanning emission electron microscopy (SEM) confirmed spherical shape and around 100 nm size of prepared formulations. Transmission electron microscopy (HRTEM) showed crystal shape of QDs with size of 2-3 nm. Drug release study obtained controlled release of encapsulated DOX and stability of formulation in physiologic condition. MTT and flow cytometry results demonstrated that AS1411 aptamer could enhance both toxicity and cellular uptake in nucleolin overexpressing cell lines (P < 0.05). Moreover, aptamer targeted formulation could increase survival rate and tumor inhibitory growth effect in 4T1 tumor baring mice (P < 0.05). Our results verify that aptamer targeted polymersomes loaded with non-toxic QDs as a diagnostic agent and DOX as an anti-cancer drug, could provide a theranostic platform with the purpose of optimization of treatment process and minimization of systemic side effects.

Modulation of intracellular ceramide using polymeric nanoparticles to overcome multidrug resistance in cancer.

Although multidrug resistance (MDR) is known to develop through a variety of molecular mechanisms within the tumor cell, many tend to converge toward the alteration of apoptotic signaling. The enzyme glucosylceramide synthase (GCS), responsible for bioactivation of the proapoptotic mediator ceramide to a nonfunctional moiety glucosylceramide, is overexpressed in many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. The purpose of this study was to investigate the therapeutic strategy of coadministering ceramide with paclitaxel, a commonly used chemotherapeutic agent, in an attempt to restore apoptotic signaling and overcome MDR in the human ovarian cancer cell line SKOV3. Poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were used to encapsulate and deliver the therapeutic agents for enhanced efficacy. Results show that indeed the cotherapy eradicates the complete population of MDR cancer cells when they are treated at their IC(50) dose of paclitaxel. More interestingly, when the cotherapy was combined with the properties of nanoparticle drug delivery, the MDR cells can be resensitized to a dose of paclitaxel near the IC(50) of non-MDR (drug sensitive) cells, indicating a 100-fold increase in chemosensitization via this approach. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is indeed due to a restoration in apoptotic signaling, although the beneficial properties of PEO-PCL nanoparticle delivery seemed to enhance the therapeutic success even further, showing the promising potential for the clinical use of this therapeutic strategy to overcome MDR.

Enzyme responsive copolymer micelles enhance the anti-biofilm efficacy of the antiseptic chlorhexidine.

Staphylococcal biofilms cause many infectious diseases and are highly tolerant to the effects of antimicrobials; this is partly due to the biofilm matrix, which acts as a physical barrier retarding the penetration and reducing susceptibility to antimicrobials, thereby decreasing successful treatment outcomes. In this study, both single and mixed micellar systems based on poly vinyl caprolactam (PCL)-polyethylene glycol (PEG) copolymers were optimised for delivery of chlorhexidine (CHX) to S. aureus, MRSA and S. epidermidis biofilms and evaluated for their toxicity using Caenorhabditis elegans. The respective polyethylene glycol (PEG) and poly vinyl caprolactam (PCL) structural components promoted stealth properties and enzymatic responsive release of CHX inside biofilms, leading to significantly enhanced penetration (56%) compared with free CHX and improving the efficacy against Staphylococcus aureus biofilms grown on an artificial dermis (2.4 log reduction of CFU). Mixing Soluplus-based micelles with Solutol further enhanced the CHX penetration (71%) and promoted maximum reduction in biofilm biomass (>60%). Nematodes-based toxicity assay showed micelles with no lethal effects as indicated by their high survival rate (100%) after 72 h exposure. This study thus demonstrated that bio-responsive carriers can be designed to deliver a poorly water-soluble antimicrobial agent and advance the control of biofilm associated infections.

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

Ethylene (ET) is the largest volume organic chemical. Mammals metabolize the olefin to ethylene oxide (EO), another important industrial chemical. The epoxide alkylates macromolecules and has mutagenic and carcinogenic properties. In order to estimate the EO burden in mice, rats, and humans resulting from inhalation exposure to gaseous ET or EO, a physiological toxicokinetic model was developed. It consists of the compartments lung, richly perfused tissues, kidneys, muscle, fat, arterial blood, venous blood, and liver containing the sub-compartment endoplasmic reticulum. Modeled ET metabolism is mediated by hepatic cytochrome P450 2E1, EO metabolism by hepatic microsomal epoxide hydrolase or cytosolic glutathione S-transferase in various tissues. EO is also spontaneously hydrolyzed or conjugated with glutathione. The model was validated on experimental data collected in mice, rats, and humans. Modeled were uptake by inhalation, wash-in-wash-out effect in the upper respiratory airways, distribution into tissues and organs, elimination via exhalation and metabolism, and formation of 2-hydroxyethyl adducts with hemoglobin and DNA. Simulated concentration-time courses of ET or EO in inhaled (gas uptake studies) or exhaled air, and of EO in blood during exposures to ET or EO agreed excellently with measured data. Predicted levels of adducts with DNA and hemoglobin, induced by ET or EO, agreed with reported levels. Exposures to 10000 ppm ET were predicted to induce the same adduct levels as EO exposures to 3.95 (mice), 5.67 (rats), or 0.313 ppm (humans). The model is concluded to be applicable for assessing health risks from inhalation exposure to ET or EO.

Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver.

Ethylene oxide (EO) is an important industrial chemical that is classified as a known human carcinogen (IARC, Group 1). It is also a metabolite of ethylene (ET), a compound that is ubiquitous in the environment and is the most used petrochemical. ET has not produced evidence of cancer in laboratory animals and is "not classifiable as to its carcinogenicity to humans" (IARC, Group 3). The mechanism of carcinogenicity of EO is not well characterized, but is thought to involve the formation of DNA adducts. EO is mutagenic in a variety of in vitro and in vivo systems, whereas ET is not. Apurinic/apyrimidinic sites (AP) that result from chemical or glycosylase-mediated depurination of EO-induced DNA adducts could be an additional mechanism leading to mutations and chromosomal aberrations. This study tested the hypothesis that EO exposure results in the accumulation of AP sites and induces changes in expression of genes for base excision DNA repair (BER). Male Fisher 344 rats were exposed to EO (100 ppm) or ET (40 or 3000 ppm) by inhalation for 1, 3 or 20 days (6h/day, 5 days a week). Animals were sacrificed 2h after exposure for 1, 3 or 20 days as well as 6, 24 and 72 h after a single-day exposure. Experiments were performed with tissues from brain and spleen, target sites for EO-induced carcinogenesis, and liver, a non-target organ. Exposure to EO resulted in time-dependent increases in N7-(2-hydroxyethyl)guanine (7-HEG) in brain, spleen, and liver and N7-(2-hydroxyethyl)valine (7-HEVal) in globin. Ethylene exposure also induced 7-HEG and 7-HEVal, but the numbers of adducts were much lower. No increase in the number of aldehydic DNA lesions, an indicator of AP sites, was detected in any of the tissues between controls and EO-, or ET-exposed animals, regardless of the duration or strength of exposure. EO exposure led to a 3-7-fold decrease in expression of 3-methyladenine-DNA glycosylase (Mpg) in brain and spleen in rats exposed to EO for 1 day. Expression of 8-oxoguanine DNA glycosylase, Mpg, AP endonuclease (Ape), polymerase beta (Pol beta) and alkylguanine methyltransferase were increased by 20-100% in livers of rats exposed to EO for 20 days. The only effects of ET on BER gene expression were observed in brain, where Ape and Pol beta expression were increased by less than 20% after 20 days of exposure to 3000 ppm. These data suggest that DNA damage induced by exposure to EO is repaired without accumulation of AP sites and is associated with biologically insignificant changes in BER gene expression in target organs. We conclude that accumulation of AP sites is not a likely primary mechanism for mutagenicity and carcinogenicity of EO.

Release of anti-restenosis drugs from poly(ethylene oxide)-poly(DL-lactic-co-glycolic acid) nanoparticles.

Dexamethasone- or rapamycin-loaded nanoparticles based on poly(ethylene oxide) and poly(dl-lactic-co-glycolic acid) block copolymers (PEO-PLGA) were prepared without additional stabilizer using the salting-out method. A fast release of drug in PBS (pH 7.4) at 37 degrees C resulting in 100% release within 5 h was observed for both drugs. The rate of drug release was substantially reduced by treating the particles with gelatin or albumin after drug loading, resulting in a linear drug release in time. It was shown that the rate of drug release is related to the amount of protein associated with the nanoparticles. After gelatin treatment of drug-loaded nanoparticles, sustained release of dexamethasone for 17 days and of rapamycin for 50 days could be achieved.

Molecular dosimetry of ethylene oxide: formation and persistence of 7-(2-hydroxyethyl)guanine in DNA following repeated exposures of rats and mice.

The formation of 7-(2-hydroxyethyl)guanine (7-HEG) in DNA of target and nontarget tissues was investigated in male B6C3F1 mice (20/group) and F344 rats (10/group) exposed to 0, 3, 10, 33, 100, or 300 (rats only) ppm ethylene oxide (ETO) by inhalation for 6 h/day for 4 weeks (5 days/week) and mice exposed to 100 ppm ETO for 1 or 3 days or 1, 2, or 4 weeks (5 days/week). The persistence of 7-HEG was studied in mice killed up to 7 days after cessation of the 4-week time-course study. In addition, the formation of O6-(2-hydroxyethyl)guanine and 3-(2-hydroxyethyl)adenine was evaluated in rats exposed to 300 ppm ETO. DNA samples from control and treated animals were analyzed for 7-HEG using neutral thermal hydrolysis, microconcentration, and high-performance liquid chromatography separation with fluorescence detection. Fluorescence-linked high-performance liquid chromatography was used for O6-(2-hydroxyethyl)guanine quantitation, and immunochromatography and gas chromatography-mass spectrometry were used for 3-(2-hydroxyethyl)adenine detection. Analysis of DNA from tissues of control mice and rats revealed the presence of peaks equivalent to 2-6 pmol 7-HEG/mg DNA. In mice exposed to 100 ppm ETO, 7-HEG accumulated to a similar extent in target and nontarget tissues, with adduct concentrations ranging from 17.5 +/- 3.0 (SE) (testis) to 32.9 +/- 1.9 (lung) pmol adduct/mg DNA after 4 weeks of exposure. Concurrent exposures of mice and rats to 100 ppm ETO for 4 weeks led to 2- to 3-fold lower concentrations of 7-HEG in mouse DNA in all tissues compared to rat DNA. 7-HEG disappeared slowly in a nearly linear fashion from the DNA of mouse kidney (t1/2 = 6.9 days) and rat brain and lung (t1/2 = 5.4-5.8 days), which was consistent with the loss of adduct mainly by chemical depurination. In contrast, a more rapid removal of 7-HEG from other mouse (t1/2 = 1.0-2.3 days) and rat (t1/2 = 2.9-4.8 days) tissues was consistent with adduct loss by depurination and DNA repair. Dose-response relationships for 7-HEG were nonlinear in both mice and rats, with the alkylating efficiency of ETO increasing at high exposures.(ABSTRACT TRUNCATED AT 400 WORDS)

cRGD-functionalized reduction-sensitive shell-sheddable biodegradable micelles mediate enhanced doxorubicin delivery to human glioma xenografts in vivo.

Biodegradable micelles are one of the most studied systems for the delivery of hydrophobic anticancer drugs. Their therapeutic efficacy in vivo is, however, suboptimal, partly due to poor tumor cell uptake as well as slow intracellular drug release. Here, we show that cRGD-functionalized intracellularly shell-sheddable biodegradable PEG-SS-PCL micelles mediate enhanced doxorubicin (DOX) delivery to U87MG glioma xenografts in vivo, resulting in significantly improved tumor growth inhibition as compared to reduction-insensitive cRGD/PEG-PCL controls. cRGD/PEG-SS-PCL micelles revealed a small size of ca. 61nm, a decent DOX loading of 14.9wt%, and triggered drug release in a reductive environment (10mM glutathione). Flow cytometry, confocal microscopy, and MTT assays demonstrated that cRGD/PEG-SS-PCL micelles with a cRGD ligand density of 20% efficiently delivered and released DOX into αvß3 integrin overexpressing U87MG cells. The in vivo pharmacokinetics studies displayed that DOX-loaded cRGD20/PEG-SS-PCL micelles had a prolonged elimination half-life time of 3.51h, which was comparable to that of cRGD20/PEG-PCL counterparts, indicating that disulfide bonds in the PEG-SS-PCL micelles are stable in the circulation. Notably, in vivo imaging and biodistribution studies in U87MG glioma xenografts showed that cRGD20/PEG-SS-PCL micelles led to efficient accumulation as well as fast drug release in the tumor. The therapeutic outcomes demonstrated that DOX-loaded cRGD20/PEG-SS-PCL micelles exhibited little side effects and superior tumor growth inhibition as compared to non-targeting PEG-SS-PCL and reduction-insensitive cRGD20/PEG-PCL counterparts. The reduction-sensitive shell-sheddable biodegradable micelles have appeared as a fascinating platform for targeted tumor chemotherapy.

Controlling the gelation of aqueous micellar solutions of ethylene-oxide-based block copoly(oxyalkylene)s.

Micellar solutions of EmPnEm copolymers may be mobile at ambient temperature and form hard gels on warming to body temperature, whereas micellar solutions of EmSnEm copolymers do not show this effect (E denotes oxyethylene, P oxypropylene and S oxyphenylethylene, and subscripts m and n denote chain lengths). The aim of this study was to combine the desirable gelation characteristics of solutions of the EmPnEm copolymers with the greater solubilising capacities of solutions of the EmSnEm copolymers. Accordingly, the gelation characteristics in aqueous solution of binary mixtures of the triblock copolymer E62P39E62 (Pluronic F87) with E137S18E137, E82S9E82 or E76S5E76 were investigated by rheological techniques. We have shown that 50/50 wt.% mixtures of E62P39E62 with either E137S18E137 or E82S9E82 at a total copolymer concentration of approximately 30 wt.% are fluids of low viscosity at temperature below 22-25 degrees C and gels of high elastic modulus at body temperature.The mixed systems have potential as vehicles for the controlled delivery of solubilised drug from gels formed in situ following subcutaneous injection of a low viscosity aqueous solution.

Monitoring occupational exposure to ethylene oxide by the determination of hemoglobin adducts.

In a study on workers in a chemical plant where ethylene oxide (EtO) is manufactured and partly used for ethylene glycol production, exposure to EtO was monitored during annual periodic health assessments in January 1988, December 1988, and March 1990 by the determination of the level of 2-hydroxyethylvaline (HOEtVal) in hemoglobin. The HOEtVal levels in workers corresponded with the potential EtO exposures. The highest level was found in December 1988, in blood samples collected 1-2 months after a shut-down, maintenance, and start-up program. The range of adduct levels found in the three examinations indicated that average EtO exposures during the 4 months preceding blood sampling were below 0.5 ppm. It was demonstrated that the method allows for the accurate monitoring of low levels of EtO exposure and provides personalized time-integrated exposure data with great discriminative power. In addition, the method may serve to identify unexpected personal exposures, which may lead to targeted exposure control measures.

Molecular dosimetry of DNA and hemoglobin adducts in mice and rats exposed to ethylene oxide.

Experiments involving ethylene oxide (ETO) have been used to support the concept of using adducts in hemoglobin as a surrogate for DNA adducts in target tissues. The relationship between repeated exposures to ETO and the formation of N-(2-hydroxyethyl)valine (HEtVal) in hemoglobin and 7-(2-hydroxyethyl)guanine (7-HEG) in DNA was investigated in male rats and mice exposed by inhalation to 0, 3, 10, 33, or 100 ppm ETO for 6 hr/day for 4 weeks, or exposed to 100 ppm (mice) or 300 ppm (rats) for 1, 3, 5, 10, or 20 days (5 days/week). HEtVal was determined by Edman degradation, and 7-HEG was quantitated by HPLC separation and fluorescence detection. HEtVal formation was linear between 3 and 33 ppm ETO and increased in slope above 33 ppm. The dose-response curves for 7-HEG in rat tissues were linear between 10 and 100 ppm ETO and increased in slope above 100 ppm. In contrast, only exposures to 100 ppm ETO resulted in significant accumulation of 7-HEG in mice. Hemoglobin adducts were lost at a greater rate than predicted by normal erythrocyte life span. The loss of 7-HEG from DNA was both species and tissue dependent, with the adduct half-lives ranging from 2.9 to 5.8 days in rat tissues (brain, kidney, liver, lung, spleen, testis) and 1.0 to 2.3 days in all mouse tissues except kidney (t1/2 = 6.9 days). The concentrations of HEtVal were similar in concurrently exposed rats and mice, whereas DNA from rats had at least 2-fold greater concentrations of 7-HEG than DNA from mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiologically based pharmacokinetic model parameter estimation and sensitivity and variability analyses for acrylonitrile disposition in humans.

A physiologically based pharmacokinetic (PBPK) model of acrylonitrile (ACN) and cyanoethylene oxide (CEO) disposition in humans was developed and is based on human in vitro data and scaling from a rat model (G. L. Kedderis et al., 1996, TOXICOL: Appl. Pharmacol.140, 422-435) for application to risk assessment. All of the major biotransformation and reactivity pathways, including metabolism of ACN to glutathione conjugates and CEO, reaction rates of ACN and CEO with glutathione and tissues, and the metabolism of CEO by hydrolysis and glutathione conjugation, were described in the human PBPK model. Model simulations indicated that predicted blood and brain ACN and CEO concentrations were similar in rats and humans exposed to ACN by inhalation. In contrast, rats consuming ACN in drinking water had higher predicted blood concentrations of ACN than humans exposed to the same concentration in water. Sensitivity and variability analyses were conducted on the model. While many parameters contributed to the estimated variability of the model predictions, the reaction rate of CEO with glutathione, hydrolysis rate for CEO, and blood:brain partition coefficient of CEO were the parameters predicted to make the greatest contributions to variability of blood and brain CEO concentrations in humans. The main contributor to predicted variance in human blood ACN concentrations in people exposed through drinking water was the Vmax for conversion of ACN to CEO. In contrast, the main contributors for variance in people exposed by inhalation were expected to be the rate of blood flow to the liver and alveolar ventilation rate, with the brain:blood partition coefficient also contributing to variability in predicted concentrations of ACN in the brain. Expected variability in blood CEO concentrations (peak or average) in humans exposed by inhalation or drinking water was modest, with a 95th-percentile individual expected to have blood concentrations 1.8-times higher than an average individual.

Quantitative estimation of the genetic risk associated with the induction of heritable translocations at low-dose exposure: ethylene oxide as an example.

This paper explores how quantitative risk assessment methods might be extended to analysis of risks to the human germ line. High inhalation exposures to ethylene oxide are reported to cause heritable translocations in male mice with a steep and nonlinear dose-response-curve. We explore quantitative estimation of risk to humans from low exposures based on these animal data, addressing questions of tissue dosimetry for this alkylating agent, expected equivalency of doses across species, germ-cell sensitivity, and extrapolation of dose-response relationship to low exposure levels. Various dose-response models are discussed in terms of their applicability to genetic end points and their ability to reflect the underlying basis of induced heritable translocations.

Concentration-response curves for ethylene-oxide-induced heritable translocations and dominant lethal mutations.

Male mice were subjected to repeated inhalation exposures to different concentrations (165, 204, 250, or 300 ppm) of ethylene oxide (EtO) during an 8.5-week period. Transmitted clastogenic effects of these exposures were measured in terms of induction of dominant lethal mutations and heritable translocations. The concentration-response curves for both endpoints are not linear but are markedly concave upward. Significant increases in dominant lethals were detected at all concentrations, except the lowest one. In comparison, the incidences of heritable translocations were significantly increased at all concentrations.

Binding of ethylene oxide in spermiogenic germ cell stages of the mouse after low-level inhalation exposure.

Mice received inhalation exposures of 3H-labeled ethylene oxide (EtO) gas at levels from 0.65 to 3.2 parts per million-hours (ppm-hr), which are below the exposure limits currently allowed for humans. Subsequently, spermatozoa were recovered from the reproductive tracts of the animals over a two-week period and assayed for the amount of bound EtO. A strong increase in the level of EtO binding occurred in late spermatid stages; these stages are also genetically sensitive to the action of EtO. The maximum binding of EtO in late spermatids amounted to 6 X 10(3) alkylations/sperm head/ppm-hr of exposure. Alkylation of the DNA within the sperm accounted for a very small fraction of the total sperm head alkylation, averaging about 20 DNA alkylations per sperm per ppm-hr of exposure over the two-week period. However, alkylation of protamine, a protein unique to sperm cells, was found to be correlated with total sperm head alkylation and accounted for nearly all of the EtO binding. Protamine alkylation appears to be a significant cause of EtO-induced genetic damage in spermiogenic cells of the mammal.