Role of environmental toxicants in the development of hypertensive and cardiovascular diseases.

The incidence of hypertension with diabetes mellitus (DM) as a co-morbid condition is on the rise worldwide. In 2000, an estimated 972 million adults had hypertension, which is predicted to grow to 1.56 billion by 2025. Hypertension often leads to diabetes mellitus that strongly puts the patients at an increased risk of cardiovascular, kidney, and/or atherosclerotic diseases. Hypertension has been identified as a major risk factor for the development of diabetes; patients with hypertension are at two-to-three-fold higher risk of developing diabetes than patients with normal blood pressure (BP). Causes for the increase in hypertension and diabetes are not well understood, environmental factors (e.g., exposure to environmental toxicants like heavy metals, organic solvents, pesticides, alcohol, and urban lifestyle) have been postulated as one of the reasons contributing to hypertension and cardiovascular diseases (CVD). The mechanism of action(s) of these toxicants in developing hypertension and CVDs is not well defined. Research studies have linked hypertension with the chronic consumption of alcohol and exposure to metals like lead, mercury, and arsenic have also been linked to hypertension and CVD. Workers chronically exposed to styrene have a higher incidence of CVD. Recent studies have demonstrated that exposure to particulate matter (PM) in diesel exhaust and urban air contributes to increased CVD and mortality. In this review, we have imparted the role of environmental toxicants such as heavy metals, organic pollutants, PM, alcohol, and some drugs in hypertension and CVD along with possible mechanisms and limitations in extrapolating animal data to humans.

Dietary Route of Exposure for Rabbit Developmental Toxicity Studies.

Dietary administration is a relevant route of oral exposure for regulatory toxicity studies of agrochemicals as it mimics potential human intake of the chemical via treated crops and commodities. Moreover, dietary administration of test compounds during a developmental toxicity study can deliver a prolonged and stable systemic exposure to the embryo or fetus at all stages of development. In this study, strategies were employed to optimize rabbit test material consumption via diet. Comparative toxicokinetic profiles of gavage versus dietary administration were evaluated in pregnant or non-pregnant New Zealand White rabbits for 2 novel agrochemicals with different plasma half-lives of elimination (sulfoxaflor, t½ = 13.5 h and halauxifen, t½ = 1 h). Dietary administration of sulfoxaflor resulted in stable 24-h plasma concentrations, whereas gavage administration resulted in a 3-fold fluctuation in plasma levels between Cmax and Cmin Dietary administration of sulfoxaflor resulted in a 2-fold higher nominal and diurnal systemic dose when compared with gavage dosing due to Cmax-related maternal toxicity following gavage. Results with the shorter half-life molecule, halauxifen, were more striking with a 6-fold diurnal fluctuation by the dietary route compared with a 368-fold fluctuation between Cmax and Cmin by gavage. Furthermore, plasma halauxifen was detectable only up to 12 h following gavage but up to 24 h following dietary administration. Finally, the presence of these compounds in fetal blood samples was demonstrated, confirming that dietary exposure is appropriate for achieving fetal exposure. Collectively, the results of these studies support the use of dietary exposure in rabbit developmental toxicity studies.

Reproductive and developmental toxicity testing: Examination of the extended one-generation reproductive toxicity study guideline.

An important aspect of safety assessment of chemicals (industrial and agricultural chemicals and pharmaceuticals) is determining their potential reproductive and developmental toxicity. A number of guidelines have outlined a series of separate reproductive and developmental toxicity studies from fertilization through adulthood and in some cases to second generation. The Extended One-Generation Reproductive Toxicity Study (EOGRTS) is the most recent and comprehensive guideline in this series. EOGRTS design makes toxicity testing progressive, comprehensive, and efficient by assessing key endpoints across multiple life-stages at relevant doses using a minimum number of animals, combining studies/evaluations and proposing tiered-testing approaches based on outcomes. EOGRTS determines toxicity during preconception, development of embryo/fetus and newborn, adolescence, and adults, with specific emphasis on the nervous, immunological, and endocrine systems, EOGRTS also assesses maternal and paternal toxicity. However, EOGRTS guideline is complex, criteria for selecting doses is unclear, and monitoring systemic dose during the course of the study for better interpretation and human relevance is not clear. This paper discusses potential simplification of EOGRTS, suggests procedures for relevant dose selection and monitors systemic dose at multiple life-stages for better interpretation of data and human relevance.

Life-stage-, sex-, and dose-dependent dietary toxicokinetics and relationship to toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) in rats: implications for toxicity test dose selection, design, and interpretation.

Life-stage-dependent toxicity and dose-dependent toxicokinetics (TK) were evaluated in Sprague Dawley rats following dietary exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D renal clearance is impacted by dose-dependent saturation of the renal organic anion transporter; thus, this study focused on identifying inflection points of onset of dietary nonlinear TK to inform dose selection decisions for toxicity studies. Male and female rats were fed 2,4-D-fortified diets at doses to 1600 ppm for 4-weeks premating, <2 weeks during mating, and to test day (TD) 71 to parental (P1) males and to P1 females through gestation/lactation to TD 96. F1 offspring were exposed via milk with continuing diet exposure until postnatal day (PND) 35. As assessed by plasma area under the curve for the time-course plasma concentration, nonlinear TK was observed ≥ 1200 ppm (63 mg/kg/day) for P1 males and between 200 and 400 ppm (14-27 mg/kg/day) for P1 females. Dam milk and pup plasma levels were higher on lactation day (LD) 14 than LD 4. Relative to P1 adults, 2,4-D levels were higher in dams during late gestation/lactation and postweaning pups (PND 21-35) and coincided with elevated intake of diet/kg body weight. Using conventional maximum tolerated dose (MTD) criteria based on body weight changes for dose selection would have resulted in excessive top doses approximately 2-fold higher than those identified incorporating critical TK data. These data indicate that demonstration of nonlinear TK, if present at dose levels substantially above real-world human exposures, is a key dose selection consideration for improving the human relevance of toxicity studies compared with studies employing conventional MTD dose selection strategies.

An F1-extended one-generation reproductive toxicity study in Crl:CD(SD) rats with 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10-12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The "No Observed Adverse Effect Level" for systemic toxicity was 300 ppm in both males (16.6 mg/kg/day) and females (20.6 mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.

TK Modeler version 1.0, a Microsoft® Excel®-based modeling software for the prediction of diurnal blood/plasma concentration for toxicokinetic use.

TK Modeler 1.0 is a Microsoft® Excel®-based pharmacokinetic (PK) modeling program created to aid in the design of toxicokinetic (TK) studies. TK Modeler 1.0 predicts the diurnal blood/plasma concentrations of a test material after single, multiple bolus or dietary dosing using known PK information. Fluctuations in blood/plasma concentrations based on test material kinetics are calculated using one- or two-compartment PK model equations and the principle of superposition. This information can be utilized for the determination of appropriate dosing regimens based on reaching a specific desired C(max), maintaining steady-state blood/plasma concentrations, or other exposure target. This program can also aid in the selection of sampling times for accurate calculation of AUC(24h) (diurnal area under the blood concentration time curve) using sparse-sampling methodologies (one, two or three samples). This paper describes the construction, use and validation of TK Modeler. TK Modeler accurately predicted blood/plasma concentrations of test materials and provided optimal sampling times for the calculation of AUC(24h) with improved accuracy using sparse-sampling methods. TK Modeler is therefore a validated, unique and simple modeling program that can aid in the design of toxicokinetic studies.

Statistical methodology to determine kinetically derived maximum tolerated dose in repeat dose toxicity studies.

Several statistical approaches were evaluated to identify an optimum method for determining a point of nonlinearity (PONL) in toxicokinetic data. (1) A second-order least squares regression model was fit iteratively starting with data from all doses. If the second order term was significant (α<0.05), the dataset was reevaluated with successive removal of the highest dose until the second-order term became non-significant. This dose, whose removal made the second order term non-significant, is an estimate of the PONL. (2) A least squares linear model was fit iteratively starting with data from all doses except the highest. The mean response for the omitted dose was compared to the 95% prediction interval. If the omitted dose falls outside the confidence interval it is an estimate of the PONL. (3) Slopes of least squares linear regression lines for sections of contiguous doses were compared. Nonlinearity was suggested when slopes of compared sections differed. A total of 33 dose-response datasets were evaluated. For these toxicokinetic data, the best statistical approach was the least squares regression analysis with a second-order term. Changing the α level for the second-order term and weighting the second-order analysis by the inverse of feed consumption were also considered. This technique has been shown to give reproducible identification of nonlinearities in TK datasets.

Response of a single 'mega intramuscular dose' of vitamin D on serum 25OHD and parathyroid hormone levels.

OBJECTIVE: To determine the changes produced in serum 25OHD and iPTH levels after 600,000 IU of injection cholecalciferol in volunteers. STUDY DESIGN: Interventional study. PLACE AND DURATION OF STUDY: Section of Chemical Pathology, Department of Pathology and Microbiology, the Aga Khan University Hospital, Karachi, from June 2009 - June 2010. METHODOLOGY: Volunteers of either gender aged 18-40 years with known 25OHD, calcium (Ca), creatinine (Cr) and phosphorous (P) levels were included in the study. Subjects on therapy like vitamin D and calcium supplements, corticosteroids or anti-epileptic medicines, primary hyperparathyroidism and hypercalcaemia, with co-morbidity like renal failure, liver disease and history of malabsorption, diarrhea or hyperthyroidism were excluded. All volunteers were given an intramuscular injection of vitamin D3 (cholecalciferol, 600,000 IU). After 8 weeks, serum 25OHD, iPTH, Ca and P levels were determined again. For 25OHD level, cut-off of ² 50 nmol/l was defined as deficient, 50-75 nmol/l as insufficient and ³ 75 as optimal level. RESULTS: Mean 25OHD and iPTH levels were 35.06 ± 16.6 nmol/l and 81.15 ± 76.78 pg/ml respectively at baseline. Seventeen volunteers were 25OHD deficient. Five had high iPTH levels (25%) (mean 156 ± 123.7 pg/ml). 25OHD and iPTH showed a significant inverse correlation at baseline (< 0.01). After 8 weeks of injection vitamin D 25OHD levels became optimal in 6 subjects (35%) [mean 92.9 ± 16.6 nmol/l]. It remained low in 5 volunteers (25%) [mean 41.6 ± 9.6 nmol/l] while insufficient levels were seen in 9 volunteers (40%) [mean 63.3±5.8 nmol/l]. Follow-up mean Ca, P and iPTH were 2.25 mmol/l (± 0.09), 1.1 (± 0.1) and 47.52 pg/ml (± 22.56) respectively. A significant increase in mean 25OHD level was seen at follow-up (p < 0.01), while the change in PTH was insignificant (p=0.05). CONCLUSION: Single mega-dose of cholecalciferol achieved optimal levels of 25OHD in 35% of subjects after eight weeks of supplementation.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.

Toxicokinetic profile of N-(2-aminoethyl)ethanolamine in the female Wistar rat and distribution into the late gestation fetus and milk.

N-(2-aminoethyl)ethanolamine (AEEA) caused aneurysms of the great vessels in rats exposed in utero and during the first days post partum, exacerbated by postnatal treatment of the lactating dams (Moore et al., 2012). The purpose of this work was to examine the systemic availability of AEEA during gestation and early lactation. The absorption of AEEA was determined following oral administration to nonpregnant and pregnant female Wistar rats. A single dose administered by gavage (0.5 or 50 mg/kg) on gestation day 18 was rapidly and extensively (>90%) absorbed from the gastrointestinal tract (absorption t(1/2) = 0.1-0.2 hr). Elimination from the plasma followed a biphasic pattern, with a rapid elimination phase (t(1/2 α) = 1.6-1.8 hr) followed by a slower phase (t(1/2 ß) = 16.7-17.3 hr). Following repeated gavage administration during gestation day 17 to 19, (14) C-AEEA-derived radioactivity readily partitioned into the fetus and was evenly distributed therein, but cleared approximately twofold slower from the fetal blood and tissues than the maternal blood and chorioallantoic placenta. When administered to lactating dams during lactation days 1 to 12, (14) C-AEEA-derived radioactivity preferentially partitioned into the milk reaching levels that were between 1.6- and 2.5-fold higher than the maternal blood. Although the concentration of AEEA equivalents in the maternal blood remained quite consistent, the concentration in the milk fell by almost 40% between lactation days 4 and 12, probably reflecting an increase in milk production over this same period. We confirm exposure of the offspring to AEEA both in utero and during lactation, but that AEEA does not appear to specifically concentrate in the great vessels.

Subchronic toxicity and genotoxicity of diiodomethyl-p-tolylsulfone (DIMPTS) in laboratory animals.

These studies were conducted to determine subchronic toxicity and genotoxicity of the biocide diiodomethyl-p-tolysulfone (DIMPTS) in rats and dogs. Male and female Sprague-Dawley rats and Beagle dogs were administered DIMPTS for 90-days via the diet at 0, 5, 20, and 80 mg/kg/day to rats and via capsules at 0, 2, 10, and 60 mg/kg/day to dogs. In rats, the only treatment-related finding was squamous metaplasia of the salivary gland duct in the 80 mg/kg/day group. In dogs, female body weights in the high-dose group were significantly lower than controls. Altered clinical pathology parameters were considered secondary to inflammatory changes observed in some of the dogs. Treatment-related alterations were found in the thyroid glands, salivary glands, GI-tract in the mid- and/or high-dose groups. DIMPTS was negative in the four in vitro and one in vivo genotoxicity assays. The toxicological effects noted in the two mammalian species are consistent with the principal toxic effects of iodine, and are proposed to arise from release of iodide from the DIMPTS molecule with toxic sequelae.

Role of iodine in the toxicity of diiodomethyl-p-tolylsulfone (DIMPTS) in rats: ADME.

Metabolism of diiodomethyl-p-tolylsulfone (DIMPTS) was investigated in rats to determine the role of iodide in its toxicity. Fischer 344 (F-344) (5 or 50mg/kg) or Sprague Dawley (SD) (5mg/kg) rats were gavaged with (14)C-DIMPTS or dermally applied with 5mg/kg (F-344 only) and absorption, distribution, metabolism and excretion (ADME) determined. Additional experiments were conducted with its deiodinated analog (methyl-p-tolylsulfone, MPTS) in female F-344 rats (20mg/kg) for comparison. Orally administered (14)C-DIMPTS was rapidly absorbed and eliminated in urine (92%). The elimination t(½) was 1-4h. Dermally applied (14)C-DIMPTS remained undetectable in plasma with bioavailability ≈ 7%, only 5-7% of the dose was recovered in urine. DIMPTS liberated one or both of its iodine atoms upon absorption. The rate of elimination of the liberated iodide from the systemic circulation was 2- to 3-fold slower in SD than F-344 rats, which resulted in higher bioavailability of iodide to SD rats. DIMPTS was primarily oxidized at the benzylic methyl moiety forming the corresponding benzoic acid. Glutathione conjugation on the sulfonyl methyl group, via displacement of I(-) was also observed. Overall 67-80% of the total iodine atoms were metabolically released from DIMPTS. The MPTS was rapidly absorbed from the GI tract, metabolized and eliminated in urine similar to that of DIMPTS. These data were compared to iodide toxicokinetic results of a reproductive toxicity study for DIMPTS (80 mg/kg/day) and MPTS (32 mg/kg/day), where DIMPTS was toxic to dams and pups, while MPTS caused no toxicity. These data show that the liberated iodide is the ultimate toxicant of DIMPTS, which is readily transported to pups through milk, while the methyltolylsulfone backbone structure (MPTS) of DIMPTS is relatively nontoxic.

Use of toxicokinetics to support chemical evaluation: Informing high dose selection and study interpretation.

Toxicokinetic (TK) information can substantially enhance the value of the data generated from toxicity testing, and is an integral part of pharmaceutical safety assessment. It is less widely used in the chemical, agrochemical and consumer products industries, but recognition of its value is growing, as reflected by increased reference to the use of TK information in new and draft OECD test guidelines. To help promote increased consideration of the important role TK can play in chemical risk assessment, we have gathered practical examples from the peer-reviewed literature, as well as in-house industry data, that highlight opportunities for the use of TK in the selection of dose levels. Use of TK can help to ensure studies are designed to be of most relevance to assessing potential risk in humans, and avoid the use of excessively high doses that could result in unnecessary suffering in experimental animals. Greater emphasis on the potential contribution of TK in guiding study design and interpretation should be incorporated in regulatory data requirements and associated guidance.

Role of iodine in diiodomethyl-p-tolylsulfone induced reproductive toxicity in rats: proposed mode of action.

The biocide diiodomethyl-p-tolylsulfone (DIMPTS) caused dystocia, decreased neonatal survival and hypothyroidism in rat reproduction studies resembling the effects caused by iodine. One molecule of DIMPTS contains two iodine moieties that are hydrolyzed upon ingestion and systemically absorbed, suggesting iodine toxicity as a probable mode of action for the effects observed in rats. This study compared the effects induced by DIMPTS and an equimolar concentration of its de-iodinated analogue, methyl-p-tolylsulfone (MPTS). Groups of 20 female Sprague Dawley rats were fed diets supplying 80 mg DIMPTS/kg/day, 32 mg MPTS/kg/day or control feed from prior to breeding through lactation and gonadal function, mating performance, conception, gestation, parturition, lactation, survival, growth and development of pups evaluated through postnatal day 7. Serum thyroid hormones and iodine levels in milk and sera were also determined. Females given DIMPTS had increased incidence of vulvar discharge and dystocia, decreased litter size, decreased body weights and feed consumption, increased thyroid weights, thyroid follicular cell hypertrophy with decreased colloid, decreased triidothyronine, and increased thyroid stimulating hormone levels. DIMPTS pups had decreased neonatal survival and body weights. These effects were associated with elevated levels of iodine in milk and sera. In contrast, MPTS did not produce similar effects in adult females or their offspring. These data support the hypothesis that the dystocia, altered neonatal survival and hypothyroidism following repeated dietary administration of DIMPTS were due to excessive iodine released from DIMPTS during absorption and metabolism.

Diiodomethyl-p-tolylsulfone: evaluation of the mode of action for reproductive toxicity.

The mode of action (MOA) underpinning the reproductive toxicity of diiodomethyl-p-tolylsulfone (DIMPTS) is excess systemic iodine levels, resulting in hypothyroidism. This MOA evaluation also addresses the potential for toxicity and adverse health outcomes during critical windows of development for different tissues. The data indicate that testicular development in the neonate represents the tissue and life-stage that are most sensitive to iodine toxicity. Life-stage specific dosimetry appears to be a major determinant of this sensitivity, with the neonate being exposed to higher levels of iodine than the fetus during the period of testicular development, in particular Sertoli cell maturation and differentiation. While no reports could be found in the literature linking excess iodine exposure in humans to testicular toxicity, there is evidence that neonates born to mothers with excessive iodine intake do exhibit signs of transient hypothyroidism. Although there are major physiological and temporal differences in testicular development and Sertoli cell replication between the rat and human, it is not inconceivable that continuous long term exposures to excess iodine first from maternal milk and then in the diet through to the onset of puberty could affect testicular development. However, exposures to iodinated substances - such as DIMPTS - contribute less than 1% of the required daily iodine intake for normal fetal and neonatal development and, consequently, continuous exposure to excess iodine during the pre-pubertal period is unlikely. As exposures to DIMPTS are both very low and sporadic in nature it is not likely that they represent any risk to health at any life-stage.

Kinetics of trihalogenated acetic acid metabolism and isoform specificity in liver microsomes.

This study determined the metabolism of 3 drinking water disinfection by-products (halogenated acetic acids [HAAs]), bromodichloroacetic acid (BDCAA), chlorodibromoacetic acid (CDBAA), and tribromoacetic acid (TBAA), using rat, mouse, human liver microsomes, and recombinant P450. Metabolism proceeded by reductive debromination forming a di-HAA; the highest under nitrogen >>2% oxygen > atmospheric headspaces. V (max) for the loss of tri-HAA was 4 to 5 times higher under nitrogen than atmospheric headspace. Intrinsic metabolic clearance was TBAA>CDBAA>>BDCAA. At the high substrate concentrations, tri-HAA consumption rate was 2 to 3 times higher than the formation of di-HAA. Liberation of Br(-) from TBAA corresponded to the expected amount produced after DBAA formation, indicating retention of Br(-) by additional metabolite/metabolites. Subsequent experiments with CDBAA detected negligible formation of chlorodibromomethane (CDBM) and failed to account for the missing tri-HAA. Carbon monoxide and especially diphenyleneiodonium ([DPI] P450 reductase inhibitor) blocked CDBAA metabolism. Other chemical inhibitors were only partially able to block CDBAA metabolism. Most effective were inhibitors of CYP 2E1 and CYP 3A4. Immunoinhibition studies using human liver microsomes and anti-human CYP 2E1 antibodies were successful in reducing CDBAA metabolism. However, CDBAA metabolism in wild-type (WT) and CYP 2E1 knockout (KO) mouse liver microsomes was similar, suggesting significant interspecies differences in CYP isoform in tri-HAA metabolism. Additional assessment of CYP isoform involvement was complicated by the finding that recombinantly expressed rat and human P450 reductase was able to metabolize CDBAA, which may be a contributing factor in interspecies differences in tri-HAA metabolism.

Dermal penetration of ethylene glycol through human skin in vitro.

This study was conducted to determine the in vitro dermal absorption of ethylene glycol (EG) through dermatomed human abdominal skin (containing epidermis and dermis), obtained from cadavers within 24 hours of death and kept frozen until processed. Three formulations of EG (neat, 50%, and 10% aqueous solutions) were applied in triplicate to skin samples from 6 donors, and placed in Teflon Bronaugh flow-through diffusion cells. Barrier integrity of each sample was evaluated with (3)H-H(2)O prior to applying EG and only data from samples passing the test were used. A physiological receptor fluid was pumped beneath the skin samples and collected in a fraction collector at predetermined time points through 24 hours. Possible volatilized EG was trapped in a charcoal basket located above each skin sample. Each skin sample was treated with an infinite dose of 500 microL of EG formulation/cm(2). At the end of 24 hours, volatilized EG trapped in the headspace was collected, the unabsorbed dose was removed from the skin and the skin was rinsed, tape stripped, and solubilized along with a rinse of the flow-through cells, and total radioactivity was determined. Only a small fraction (

In vitro metabolism and covalent binding of ethylbenzene to microsomal protein as a possible mechanism of ethylbenzene-induced mouse lung tumorigenesis.

This study was conducted to determine species differences in covalent binding of the reactive metabolites of ethylbenzene (EB) formed in the liver and lung microsomes of mouse, rat and human in the presence of NADPH. These data further the understanding of the mechanism by which EB causes mouse specific lung toxicity and a follow-up to our earlier report of the selective elevation, although minor, of the ring-oxidized reactive metabolites in mouse lung microsomes (Saghir et al., 2009). Binding assays were also conducted with or without 5-phenyl-1-pentyne (5P1P), an inhibitor of CYP 2F2, and diethyldithiocarbamate (DDTC), an inhibitor of CYP 2E1 to evaluate their role in the formation of the related reactive metabolites. Liver and lung microsomes were incubated with (14)C-EB (0.22 mM) in the presence of 1mM NADPH under physiological conditions for 60 min. In lung microsomes, binding activity was in the order of mouse (812.4+/-102.2 pmol/mg protein)>>rat (57.0+/-3.2 pmol/mg protein). Human lung microsomes had little binding activity (15.7+/-1.4 pmol/mg protein), which was comparable to the no-NADPH control (9.9-16.7 pmol/mg protein). In liver microsomes, mouse had the highest activity (469.0+/-38.5 pmol/mg protein) followed by rat (148.3+/-14.7 pmol/mg protein) and human (89.8+/-3.0 pmol/mg protein). Presence of 5P1P or DDTC decreased binding across species and tissues. However, much higher inhibition was observed in mouse (86% [DDTC] and 89% [5P1P]) than rat (56% [DDTC] and 59% [5P1P]) lung microsomes. DDTC showed approximately 2-fold higher inhibition of binding in mouse and human liver microsomes than 5P1P (mouse=85% vs. 40%; human=59% vs. 36%). Inhibition in binding by DDTC was much higher (10-fold) than 5P1P (72% vs. 7%) in rat liver microsomes. These results show species, tissue and enzyme differences in the formation of reactive metabolites of EB. In rat and mouse lung microsomes, both CYP2E1 and CYP2F2 appear to contribute in the formation of reactive metabolites of EB. In contrast, CYP2E1 appears to be the primary CYP isozyme responsible for the reactive metabolites of EB in the liver.

Mechanism of ethylbenzene-induced mouse-specific lung tumor: metabolism of ethylbenzene by rat, mouse, and human liver and lung microsomes.

This study was conducted to determine species differences in the metabolism of ethylbenzene (EB) in liver and lung. EB (0.22-7.0mM) was incubated with mouse, rat and human liver and lung microsomes and the formation of 1-phenylethanol (1PE), acetophenone (AcPh), 2-ethylphenol (2EP), 4-ethylphenol (4EP), 2,5-ethylquinone, and 3,4-ethylquinone were measured. Reactive metabolites (2,5-dihydroxyethylbenzene-GSH [2EP-GSH] and 3,4-dihydroxyethylbenzene-GSH [4EP-GSH]) were monitored via glutathione (GSH) trapping technique. None of the metabolites were formed at detectable levels in incubations with human lung microsomes. Percent conversion of EB to 1PE ranged from 1% (rat lung; 7.0mM EB) to 58% (mouse lung; 0.22 mM EB). More 1PE was formed in mouse lung than in mouse liver microsomes, although formation of 1PE by rat liver and lung microsomes was similar. Metabolism of EB to 1PE was in the order of mouse > rat > human. Formation of AcPh was roughly an order of magnitude lower than 1PE. Conversion of EB to ring-hydroxylated metabolites was much lower (0.0001% [4EP-GSH; rat lung] to 0.6% [2EP-GSH; mouse lung]); 2EP-GSH was typically 10-fold higher than 4EP-GSH. Formation of 2EP-GSH was higher by lung (highest by mouse lung) than liver microsomes and the formation of 2EP-GSH by mouse liver microsomes was higher than rat and human liver microsomes. Increasing concentrations of EB did lead to a decrease in amount of some formed metabolites. This may indicate some level of substrate- or metabolite-mediated inhibition. High concentrations of 2EP and 4EP were incubated with microsomes to further investigate their oxidation to ethylcatechol (ECat) and ethylhydroquinone (EHQ). Conversion of 2EP to EHQ ranged from 6% to 9% by liver (mouse > human > rat) and from 0.1% to 18% by lung microsomes (mouse >> rat >> human). Conversion of 4EP to ECat ranged from 2% to 4% by liver (mouse > human approximately rat) and from 0.3% to 7% by lung microsomes (mouse >> rat >> human). Although ring-oxidized metabolites accounted for a relatively small fraction of overall EB metabolism, its selective elevation in mouse lung microsomes is nonetheless consistent with the hypothesized mode of action for observed preferential toxicity of EB to the lung in this species.

Determination of ADME and bioavailability following intravenous, oral, and dermal routes of exposure.

Humans are exposed to chemicals either voluntarily or involuntarily through several routes. Therapeutic drugs are introduced into the human system via a number of routes including, but not limited to, oral, inhalation, intravenous (i.v.), topical, and subcutaneous. For occupational and environmental chemicals, the major routes of human exposure are inhalation, dermal, and oral. To determine the extent of exposure to chemicals, the concentration of the active molecules is measured in a biological medium. Determination of absolute and/or relative bioavailability of occupational and environmental chemical exposure through different routes is critical in understanding the risk to the general population of a low-level exposure to these chemicals. This unit describes typical protocol designs to generate data for the calculation of absorption, distribution, metabolism, and elimination (ADME) and absolute and relative bioavailability of chemicals when exposed through i.v., oral, and dermal routes.