Discovery of the cancer cell selective dual acting anti-cancer agent (Z)-2-(1H-indol-3-yl)-3-(isoquinolin-5-yl)acrylonitrile (A131).

Selective targeting of cancer cells over normal cells is a key objective of targeted therapy. However few approaches achieve true mechanistic selectivity resulting in debilitating side effects and dose limitation. In this work we describe the discovery of A131 (4a), a new agent with an unprecedented dual mechanism of action targeting both mitosis and autophagy. Compound 4a was first identified in a phenotypic screen in which HeLa cells treated with 4a manifested mitotic arrest along with formation of multiple vesicles. Further investigations showed that 4a causes an increase in mitotic marker pH3 and autophagy marker LC3. Importantly 4a induces cell death in cancer cells while sparing normal cells which regrow after 4a is removed. Dual activities against pH3 and LC3 markers are required for cancer cell selectivity. An extensive SAR investigation confirmed 4a as the optimal dual inhibitor with potency against a panel of 30 cancer cell lines (average antiproliferative GI50 1.5 µM). In a mouse model of paclitaxel-resistant colon cancer, 4a showed 74% tumor growth inhibition when administered at a dose of 20 mg/kg IP twice a day.

Uncertainty and variability in the exposure reconstruction of chemical incidents--the case of acrylonitrile.

The application of human physiologically based pharmacokinetic (PBPK) modeling combined with measured biomonitoring data, has a great potential to backtrack external exposure to chemicals during chemical incidents. So far, an important shortcoming of 'reversed dosimetry' is that uncertainty and variability in the model predictions are often neglected. The aim of this paper is to characterize the variation in predicted environmental air concentrations by means of reversed dosimetry as a result of uncertainty in chemical-specific input data and variability in physiological parameters. Human biomonitoring data (N-2-cyanoethylvaline in blood) from a chemical incident with acrylonitrile (ACN) combined with the BioNormtox PBPK model are used as a case to reconstruct the air concentration and uncertainty thereof at the time of the incident. The influence of uncertainty in chemical-specific properties and exposure duration, and interindividual variability in physiological parameters on the reconstructed air exposure concentrations were quantified via nested Monte Carlo simulation. The range in the reconstructed air concentrations of ACN during the incident was within a factor of 3. Uncertainty in the exact exposure duration directly after the chemical accident was found to have a dominant influence on the model outcomes. It was also shown that uncertainty can be further reduced by collecting human biomonitoring data as soon as possible after the incident. Finally, the collection of specific information about individual physiological parameters from the victims, such as body weight, may further reduce the variation by 5 to 20% in our case study. Future research should include the comparison of reversed dosimetry model outcomes with measured air and biological concentrations to further increase the confidence in the model approach and its implementation in practice.

(Z)-2-(2-bromophenyl)-3-{[4-(1-methyl-piperazine)amino]phenyl}acrylonitrile (DG172): an orally bioavailable PPARß/δ-selective ligand with inverse agonistic properties.

The ligand-regulated nuclear receptor peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a potential pharmacological target due to its role in disease-related biological processes. We used TR-FRET-based competitive ligand binding and coregulator interaction assays to screen 2693 compounds of the Open Chemical Repository of the NCI/NIH Developmental Therapeutics Program for inhibitory PPARß/δ ligands. One compound, (Z)-3-(4-dimethylamino-phenyl)-2-phenyl-acrylonitrile, was used for a systematic SAR study. This led to the design of derivative 37, (Z)-2-(2-bromophenyl)-3-{[4-(1-methyl-piperazine)amino]phenyl}acrylonitrile (DG172), a novel PPARß/δ-selective ligand showing high binding affinity (IC(50) = 27 nM) and potent inverse agonistic properties. 37 selectively inhibited the agonist-induced activity of PPARß/δ, enhanced transcriptional corepressor recruitment, and down-regulated transcription of the PPARß/δ target gene Angptl4 in mouse myoblasts (IC(50) = 9.5 nM). Importantly, 37 was bioavailable after oral application to mice with peak plasma levels in the concentration range of its maximal inhibitory potency, suggesting that 37 will be an invaluable tool to elucidate the functions and therapeutic potential of PPARß/δ.

Blood concentrations of acrylonitrile in humans after oral administration extrapolated from in vivo rat pharmacokinetics, in vitro human metabolism, and physiologically based pharmacokinetic modeling.

The present study defined a simplified physiologically based pharmacokinetic (PBPK) model for acrylonitrile in humans based on in vitro metabolic parameters determined using relevant liver microsomes, coefficients derived in silico, physiological parameters derived from the literature, and a prior previously developed PBPK model in rats. The model basically consists of a chemical absorption compartment, a metabolizing compartment, and a central compartment for acrylonitrile. Evaluation of a previous rat model was performed by comparisons with experimental pharmacokinetic values from blood and urine obtained from rats in vivo after oral treatment with acrylonitrile (30 mg/kg, a no-observed-adverse-effect level) for 14 days. Elimination rates of acrylonitrile in vitro were established using data from rat liver microsomes and from pooled human liver microsomes. Acrylonitrile was expected to be absorbed and cleared rapidly from the body in silico, as was the case for rats confirmed experimentally in vivo with repeated low-dose treatments. These results indicate that the simplified PBPK model for acrylonitrile is useful for a forward dosimetry approach in humans. This model may also be useful for simulating blood concentrations of other related compounds resulting from exposure to low chemical doses.

Derivation of noncancer reference values for acrylonitrile.

Dose-response assessments were conducted for the noncancer effects of acrylonitrile (AN) for the purposes of deriving subchronic and chronic oral reference dose (RfD) and inhalation reference concentration (RfC) values. Based upon an evaluation of available toxicity data, the irritation and neurological effects of AN were determined to be appropriate bases for deriving reference values. A PBPK model, which describes the toxicokinetics of AN and its metabolite 2-cyanoethylene oxide (CEO) in both rats and humans, was used to assess the dose-response data in terms of an internal dose measure for the oral RfD values, but could not be used in deriving the inhalation RfC values. Benchmark dose (BMD) methods were used to derive all reference values. Where sufficient information was available, data-derived uncertainty factors were applied to the points of departure determined by BMD methods. From this assessment, subchronic and chronic oral RfD values of 0.5 and 0.05 mg/kg/day, respectively, were derived. Similarly, subchronic and chronic inhalation RfC values of 0.1 and 0.06 mg/m(3), respectively, were derived. Confidence in the reference values derived for AN was considered to be medium to high, based upon a consideration of the confidence in the key studies, the toxicity database, dosimetry, and dose-response modeling.

Neurobehavioral alterations in rats exposed to acrylonitrile in drinking water.

This study was carried out on rodents, to explore the neurobehavioral effects of acrylonitrile (AN) administered in drinking water. Thirty, male, Sprague-Dawley rats were randomly divided into three groups: two exposure groups (50 and 200 ppm AN), and one control group (tap water without AN). Three tests, including the open field test, rotarod test and spatial water maze, were applied to evaluate locomotor activities, motor co-ordination and learning and memory, respectively, prior to initiation of the treatment, and at Week 4, 8 and 12 postexposure. There were no consistent changes in the open field test, except for locomotion and grooming episodes. In the rotarod test, AN significantly decreased the latencies to fall in a dose and time-dependent manner. In the spatial water maze test, rats exposed to AN for 12 weeks had significantly more training times and longer escape latencies than control animals. These findings indicate that oral exposure to AN induces neurobehavioral alterations in rats.

Report of an independent peer review of an acrylonitrile risk assessment.

A peer review panel made up of experts in toxicology, epidemiology, cancer mode of action (MOA), cancer mechanisms, carcinogenicity, genotoxicity, dose-response, US Environmental Protection Agency (EPA) cancer and noncancer methods, pharmacokinetic modeling and acrylonitrile, met on 22-23 September 2003 in Cincinnati, OH. The purpose of the meeting was to provide an independent review of a risk assessment of acrylonitrile that had been prepared by the Acrylonitrile Group (AN Group). Toxicology Excellence for Risk Assessment (TERA) organized the peer review and selected the panel. The panel discussed the toxicity and epidemiology literature of acrylonitrile and MOA information, and reached conclusions regarding its MOA, weight of evidence (WOE) for carcinogenicity, preferred approach for dose-response assessment and risk values. This paper summarizes the discussion and conclusions of the panel regarding the acrylonitrile assessment. Subsequent to the peer review, the authors of the acrylonitrile assessment revised their report and the panel reviewed the revised report. A manuscript of the revised assessment is being published in Regulatory Toxicology and Pharmacology.

On the importance of exposure variability to the doses of volatile organic compounds.

The connection between occupational exposure to volatile organic compounds (VOCs) and the resulting internal doses is complicated by variability in air levels from day to day and by nonlinear kinetics of metabolism. We investigated long-term liver doses of VOCs and their metabolites using a physiologically based toxicokinetic model, to which 10,000 random 8-h exposures were inputted. Three carcinogenic VOCs were studied (i.e., benzene, perchloroethylene, and acrylonitrile); these compounds are all bioactivated in the liver and represent a wide range of an important toxicokinetic parameter Vmax/QL x KM. For each VOC, simulations were performed using mean air concentrations (muX) between 0.0003 and 1 mg/l (which covers both linear and saturated metabolism) and using coefficients of variation of exposure (CVX) between 0.23 and 2.18 (which includes most occupational settings). Two long-term measures of internal dose were examined, i.e., the area under the liver concentration-time curve (AUCL) and the area under the metabolic rate-time curve (AURC). Interestingly, both AUCL and AURC were linear functions of cumulative exposure (CE, mg x h/l air) even when metabolism was saturated and CVX was large. Yet, at a given CE, both AUCL and AURC were affected by CVX, with the magnitude of the effect increasing with Vmax/QL x KM (i.e., perchloroethylene < benzene < acrylonitrile). Nonetheless, the effects of CVX were typically only a few percent and should be of little consequence unless a VOC has large values of Vmax/QL x KM, muX,and CVX. We conclude that CE should be a sufficient predictor of the dose of either the parent chemical (VOC) or its metabolite in the liver, even when metabolism is nonlinear. We also observed that AUCL and AURC were sensitive to changes in values of model parameters in the high-variability scenarios, suggesting that (when CVX is large) the population variability of AUCL and AURC can be quite large at a fixed CE.

Species difference in the disposition of acrylonitrile: quantitative whole-body autoradiographic study in rats and mice.

Previous studies from this and other laboratories have indicated the role of species difference in acrylonitrile (VCN) toxicity and its metabolism to cyanide. Our recent studies also indicated a more pronounced elimination of VCN following oral as compared to i.v. administration. To further characterize the mechanism of these differences on the distribution of VCN, quantitative whole-body autoradiographic distribution and elimination studies were conducted at various time points (0.08, 8, 24, 48 h) following the administration of an equivalent i.v. dose of 2-[14C]-VCN to male Fischer rats and male CD-1 mice. Whole-body autoradiographs obtained from freeze-dried and acid-extracted sections of rats and mice demonstrated a rapid uptake of 14C in liver, lungs, spleen and bone marrow at early time intervals. Quantitatively, the uptake, retention and covalent interaction of 14C were higher in organs of rats as compared to mice, over 48 h. Mice eliminated 74% of the total administered dose of 2-[14C]-VCN (expired air 4%, urine 16% and feces 54%), while rats eliminated only 26% of the dose (expired air 2%, urine 4% and feces 20%). Species differences in VCN toxicity seem to be correlated with its rate of elimination. The distribution and elimination data demonstrated that mice divest VCN more rapidly than rats. The study also demonstrated that administration of VCN in rats resulted in covalent interactions and retention of 2-[14C]-VCN/metabolites in the tissues thus exerting more chronic toxicity to rats than to mice.

Differential metabolism of acrylonitrile to cyanide is responsible for the greater sensitivity of male vs female mice: role of CYP2E1 and epoxide hydrolases.

Acrylonitrile (AN) is a potent toxicant and a known rodent carcinogen. AN epoxidation to cyanoethylene oxide (CEO) via CYP2E1 and its subsequent metabolism via epoxide hydrolases (EH) to yield cyanide is thought to be responsible for the acute toxicity and mortality of AN. Recent reports showed that male mice are more sensitive than females to the acute toxicity/mortality of AN. The present work was undertaken to assess the metabolic and enzymatic basis for the greater sensitivity of male vs female mice to AN toxicity. Male and female wild-type and CYP2E1-null mice received AN at 0, 2.5, 10, 20, or 40 mg/kg by gavage. Cyanide concentrations were measured at 1 or 3 h after dosing. Current data demonstrated that cyanide levels in blood and tissues of AN-treated wild-type mice of both sexes were significantly greater than in vehicle-treated controls and increased in a dose-dependent manner. In contrast, cyanide levels in AN-treated CYP2E1-null mice were not statistically different from those measured in vehicle-treated controls. Furthermore, higher levels of cyanide were detected in male wild-type mice vs females in association with greater sensitivity of males to the acute toxicity/mortality of this chemical. Using Western blot analysis, negligible difference in CYP2E1 expression with higher levels of soluble and microsomal EH (sEH and mEH) was detected in the liver of male vs female mice. In kidneys, male mice exhibited higher expression of both renal CYP2E1 and sEH than did female mice. In conclusion, higher blood and tissue cyanide levels are responsible for the greater sensitivity of male vs female mice to AN. Further, higher expression of CYP2E1 and EH in male mice may contribute to greater formation of CEO and its subsequent metabolism to yield cyanide, respectively.

Effect of route of administration on the disposition of acrylonitrile: quantitative whole-body autoradiographic study in rats.

Previous studies on the effect of route of administration on acrylonitrile (VCN) toxicity in animal models indicated high rates of metabolism in rats that received an oral dose than those received an i.p. VCN dose. To evaluate the role of route of administration on the distribution of VCN, a quantitative whole-body autoradiography (QWBA) and elimination studies were conducted. Equimolar doses of 2-[14C]-VCN were administered i.v. or p.o. to male Fischer (F-344) rats. Time course of QWBA indicated a higher retention and covalent interaction of radioactivity in the liver, spleen and bone marrow of rats received an i.v. dose of 2-14C-VCN than those received a p.o. dose. Unlike rats that received an i.v. dose of VCN, the animals received an oral dose showed a high retention of 14C in blood, stomach and gastric mucosa. Differences were also reflected in 14C elimination in urine, feces and expired air. Animals treated orally with 2-[14C]-VCN excreted 61% of the administered radioactive dose (4% in expired air, 4% in urine and 53% in feces). Rats that received an i.v. dose of 2-[14C]-VCN, however, eliminated only 30% of the total radioactive dose (expired air 2%, urine 8% and feces 21%). The results indicate that metabolism, detoxication and elimination of VCN are more pronounced following oral administration as compared to i.v. route of administration. The study also demonstrates that the systemic administration of VCN enhances its covalent interaction and retention in the tissues thus cause more toxicity in these organs.

A first approach to estimate the internal exposure to acrylamide in smoking and non-smoking adults from Germany.

Since the formation of acrylamide (AA) in the heating process of starch-containing food could be demonstrated and high contents of this substance were found in commercial food products, there is a great discussion about the possible human health risks connected with this dietary exposure. In order to determine the body burden of the general population in Germany caused by this uptake, we investigated the internal exposure to acrylamide and acrylonitrile in a group of 72 persons using haemoglobin adducts as parameters of biochemical effects. The collective was subdivided into non-smokers and smokers basing on the results of the smoking-specific acrylonitrile adduct (N-cyanoethylvaline, CEV). The median value for the adduct of AA (N-2-carbamoylethylvaline, AAV) in 25 non-smokers was 21 pmol/g globin (approximately 0.6 microgram/l blood) with a 95 percentile of 46 pmol/g globin (approximately 1.3 micrograms/l) (LOD: 12 pmol/g globin). The median level for AAV in smokers (n = 47) was found to be 85 pmol/g globin (approximately 2.3 micrograms/l blood) with a 95 percentile of 159 pmol/g globin (approximately 4.3 micrograms/l blood). Based on these results about 60 micrograms AA/d are taken up by adult non-smoking persons. According to calculations of WHO and US EPA this background exposure would lead to a cancer risk between 6 x 10(-4) and 3.6 x 10(-3). Our results confirm a body burden to AA even in persons from the non-smoking general population in Germany that is most probably caused by dietary uptake. Smoking habits considerably contribute to the level of this adduct.

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.

Polymorphism of glutathione S-transferases and susceptibility to acrylonitrile and dimethylsulfate in cases of intoxication.

In this paper, the difficulties of genetic screening of occupationally exposed subjects for the evaluation of retrospective, and prospective, health risk assessments is illustrated with reference to glutathione S-transferase (GST) function. Individual differences in the magnitude and half-life of adduct levels, derived from background and occupational exposure, are observed largely independently of genetically determined conjugator status. During detoxification, GSTs play a critical role in providing protection against electrophiles and products of oxidative stress. GSTs are a superfamily of enzymes that may have broad and overlapping substrate specificities. Deficiencies of GST isoenzymes may be compensated by the presence of other isoforms and by the use of alternative metabolic pathways. This may be one reason for the abundance of controversial data on GST polymorphisms and adverse health effects.

Acrylonitrile is a multisite carcinogen in male and female B6C3F1 mice.

Acrylonitrile is a heavily produced unsaturated nitrile, which is used in the production of synthetic fibers, plastics, resins, and rubber. Acrylonitrile is a multisite carcinogen in rats after exposure via gavage, drinking water, or inhalation. No carcinogenicity studies of acrylonitrile in a second animal species were available. The current studies were designed to assess the carcinogenicity of acrylonitrile in B6C3F1 mice of both sexes. Acrylonitrile was administered by gavage at 0, 2.5, 10, or 20 mg/kg/day, 5 days per week, for 2 years. Urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine were measured as markers of exposure to acrylonitrile. In general, there were dose-related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations in all dosed groups of mice and at all time points. Survival was significantly (p < 0.001) reduced in the top dose (20 mg/kg) group of male and female mice relative to controls. The incidence of forestomach papillomas and carcinomas was increased in mice of both sexes in association with an increase in forestomach epithelial hyperplasia. The incidence of Harderian gland adenomas and carcinomas was also markedly increased in the acrylonitrile-dosed groups. In female mice, the incidence of benign or malignant granulosa cell tumors (combined) in the ovary in the 10 mg/kg dose group was greater than that in the vehicle control group, but because of a lack of dose response, this was considered an equivocal finding. In addition, the incidences of atrophy and cysts in the ovary of the 10 and 20 mg/kg dose groups were significantly increased. The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in female mice treated with acrylonitrile at 10 mg/kg/day for 2 years. This was also considered an equivocal result. In conclusion, these studies demonstrated that acrylonitrile causes multiple carcinogenic effects after gavage administration to male and female B6C3F1 mice for 2 years.

Toxicology and carcinogenesis studies of acrylonitrile (CAS No. 107-13-1) in B6C3F1 mice (gavage studies).

UNLABELLED: Acrylonitrile is used in the production of acrylic and modacrylic fiber, elastomers, acrylonitrile-butadiene-styrene and styrene-acrylonitrile resins, nitrile rubbers, gas barrier resins, and chemical intermediates such as adiponitrile and acrylamide. Acrylonitrile was nominated for study by the National Institute of Environmental Health Sciences because of its potential for human exposure, its classification as a probable human carcinogen, evidence of its carcinogenicity in rats, and the lack of carcinogenicity studies in a second animal species. Male and female B6C3F1 mice received acrylonitrile (greater than 99% pure) in deionized water by gavage for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse peripheral blood erythrocytes. 14-WEEK STUDY: Groups of 10 male and 10 female mice were administered 0, 5, 10, 20, 40, or 60 mg acrylonitrile/kg body weight in deionized water by gavage, 5 days per week, for 14 weeks. All male and nine female mice in the 60 mg/kg groups and eight male and three female mice in the 40 mg/kg groups died on the first day of the study. The mean body weight gain of 20 mg/kg males was less than that of the vehicle control group. Clinical findings included lethargy and abnormal breathing in the 40 mg/kg groups. Leukocyte and lymphocyte counts were decreased in 20 mg/kg males and 40 mg/kg females, and a minimal hemolytic anemia was observed in 40 mg/kg females. Heart weights of 20 mg/kg males were significantly greater than those of the vehicle controls, and left cauda epididymis weights of 10 and 20 mg/kg males were significantly increased. The incidences of chronic active inflammation and hyperplasia in the forestomach of 40 mg/kg females were significantly increased. 2-YEAR STUDY: Groups of 50 male and 50 female mice were administered acrylonitrile in deionized water by gavage at doses of 0, 2.5, 10, or 20 mg/kg, 5 days per week, for 104 to 105 weeks. Urine from five male and five female mice from each group was collected at 2 weeks and at 3, 12, and 18 months and analyzed for thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations as markers of exposure to acrylonitrile. Survival, Body Weights, and Urinary Metabolite Analyses Survival of 20 mg/kg mice was significantly less than that of the vehicle control groups. Mean body weights of 20 mg/kg males and females were generally less than those of the vehicle controls throughout most of the study. Dose-related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations occurred in all dosed groups at 2 weeks and at 3, 12, and 18 months. Pathology Findings The incidences of squamous cell papilloma, squamous cell carcinoma, and squamous cell papilloma or carcinoma (combined) of the forestomach occurred with positive trends in males and females, and were present in 50% or greater of mice administered 10 or 20 mg/kg. The incidences of mild focal or multifocal epithelial hyperplasia (combined) of the forestomach in 20 mg/kg males and females and of mild diffuse or focal hyperkeratosis (combined) in 20 mg/kg males were increased. The incidences of harderian gland adenoma and adenoma or carcinoma (combined) were significantly increased in all dosed groups of males and in 10 and 20 mg/kg females, and the incidence of harderian gland hyperplasia was significantly increased in 10 mg/kg males. The incidence of benign or malignant granulosa cell tumor (combined) in the ovary of 10 mg/kg females was greater than that in the vehicle controls. The incidences of atrophy and cyst in the ovary of 10 and 20 mg/kg females were significantly increased. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in 10 mg/kg females was significantly increased. GENETIC TOXICOLOGY: Acrylonitrile was mutagenic in S. typhimurium strains TA100 and TA1535 in the presence of S9 liver enzymes; it was not mutagenic without S9 activation in these two strains. No mutagenic activity was observed in strain TA97 or TA98 with or without S9. Acrylonitrile was mutagenic in mouse lymphoma L5178Y cells in the absence of S9; it was not tested with S9. In cultured Chinese hamster ovary cells, acrylonitrile induced sister chromatid exchanges with and without S9; chromosomal aberrations were significantly increased in the presence of S9 only. Tests for induction of sex-linked recessive lethal mutations in germ cells of male D. melanogaster were negative when acrylonitrile was administered in feed or by injection. A test for induction of reciprocal translocations in male D. melanogaster was negative. In contrast to the induction of chromosomal damage by acrylonitrile in mammalian cells in vitro, no increase in the frequency of micronucleated normochromatic erythrocytes was observed in peripheral blood samples from male or female mice administered acrylonitrile by gavage for 14 weeks. In summary, acrylonitrile induced genetic damage in vitro in bacterial and mammalian cells, but in vivo test results in D. melanogaster and in mice were negative. CONCLUSIONS: Under the conditions of this 2-year gavage study, there was clear evidence of carcinogenic activity of acrylonitrile in male and female B6C3F1 mice based on increased incidences of forestomach and harderian gland neoplasms. Neoplasms of the ovary and lung in female mice may have been related to administration of acrylonitrile. Nonneoplastic lesions of the forestomach and harderian gland in males and of the forestomach and ovary in females were associated with administration of acrylonitrile by gavage for 2 years.

Improving cancer dose-response characterization by using physiologically based pharmacokinetic modeling: an analysis of pooled data for acrylonitrile-induced brain tumors to assess cancer potency in the rat.

Historically, U.S. regulators have derived cancer slope factors by using applied dose and tumor response data from a single key bioassay or by averaging the cancer slope factors of several key bioassays. Recent changes in U.S. Environmental Protection Agency (EPA) guidelines for cancer risk assessment have acknowledged the value of better use of mechanistic data and better dose-response characterization. However, agency guidelines may benefit from additional considerations presented in this paper. An exploratory study was conducted by using rat brain tumor data for acrylonitrile (AN) to investigate the use of physiologically based pharmacokinetic (PBPK) modeling along with pooling of dose-response data across routes of exposure as a means for improving carcinogen risk assessment methods. In this study, two contrasting assessments were conducted for AN-induced brain tumors in the rat on the basis of (1) the EPA's approach, the dose-response relationship was characterized by using administered dose/concentration for each of the key studies assessed individually; and (2) an analysis of the pooled data, the dose-response relationship was characterized by using PBPK-derived internal dose measures for a combined database of ten bioassays. The cancer potencies predicted for AN by the contrasting assessments are remarkably different (i.e., risk-specific doses differ by as much as two to four orders of magnitude), with the pooled data assessments yielding lower values. This result suggests that current carcinogen risk assessment practices overestimate AN cancer potency. This methodology should be equally applicable to other data-rich chemicals in identifying (1) a useful dose measure, (2) an appropriate dose-response model, (3) an acceptable point of departure, and (4) an appropriate method of extrapolation from the range of observation to the range of prediction when a chemical's mode of action remains uncertain.

Biological markers of acute acrylonitrile intoxication in rats as a function of dose and time.

Three markers of acute acrylonitrile (AN) intoxication, namely, tissue glutathione (GSH), tissue cyanide (CN), and covalent binding to tissue protein, were studied as a function of dose and time. Doses administered and responses expected were 20 mg/kg (LD0), 50 mg/kg (LD10), 80 mg/kg (LD50), and 115 mg/kg (LD90). Liver GSH was the most sensitive marker of AN exposure. At 80 mg/kg AN, virtually complete depletion of liver GSH was observed within 30 min with no recovery through 120 min. Kidney GSH showed a similar, but less intense depletion; while blood and brain GSH were more refractory to AN. Whole blood and brain CN rose progressively during the first 60 min in a dose-dependent fashion. At the lowest dose, CN levels decreased thereafter, whereas, at the three higher doses, CN levels were maintained or continued to increase through 120 min. At the highest dose, blood and brain CN remained at acutely toxic levels through 240 min. Covalent binding increased rapidly in all tissues during the first 30 min at all doses. At the lowest dose, little additional covalent binding was observed beyond 30 min, while at the three higher doses, covalent binding increased, although at a slower rate. The data indicate that these three biologic markers of acute AN intoxication respond dramatically in a time-dependent manner in the toxic dosage range. Furthermore, the data provide evidence that AN toxicity is gated by GSH depletion in liver with the resultant termination of AN detoxification.

Dose dependence of covalent binding of acrylonitrile to tissue protein and globin in rats.

The dose dependence of acrylonitrile (AN) covalent binding to tissue protein, following a single acute exposure over a 100-fold range in dose, was measured. Covalent binding was a linear function of AN dose in the lower dose range (0.02-0.95 mmol AN/kg). The slopes of the dose-response curves indicated that tissues varied by nearly 10-fold in their reactivity with AN. The relative order of covalent binding was as follows: blood > > kidney = liver > forestomach = brain > glandular stomach > > muscle. Similar dose-response behavior was observed for globin total covalent binding and for globin N-(2-cyanoethyl) valine (CEValine) adduct formation. The latter adduct was found to represent only 0.2% of the total AN adduction to globin. Regression of tissue protein binding versus globin total covalent binding or globin CEValine adduct indicated that both globin biomarkers could be used as surrogates to estimate the amount of AN bound to tissue protein. At higher AN doses, above approximately 1 mmol/kg, a sharp break in the covalent binding dose-response curve was observed. This knot value is explained by the nearly complete depletion of liver glutathione and the resultant termination of AN detoxification. The toxicity of AN is known to increase sharply above this dose. The data suggest that a comparison of specific tissue proteins labeled by AN above and below this threshold dose may provide some insight into the mechanism of AN-induced toxicity.