Mode-of-action-based dosimeters for interspecies extrapolation of vinyl acetate inhalation risk.

Vinyl acetate is used in the manufacture of many polymers. The Clean Air Act Amendments of 1990 require that an inhalation risk assessment be conducted to assess risks to human health from ambient exposures. Vinyl acetate is a nasal carcinogen in rats and induces olfactory degeneration in rats and mice. Because of the many unique aspects of the rodent nasal cavity compared to that of humans, conventional means for extrapolating dosimetry between species are not appropriate. Physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PD) modeling can address many of these unique aspects. A PBPK/PD model has been developed for vinyl acetate, but the choice of appropriate dosimeter(s) to use for interspecies extrapolation depends on a hypothesis regarding mode of action. This article summarizes the key studies that formulate a mode of action hypothesis for vinyl acetate. Dose-response relationships for vinyl acetate-induced nonneoplastic and neoplastic responses are highly nonlinear, suggesting complex kinetic processes. Carboxylesterase-dependent metabolism of vinyl acetate forms acetic acid, a potent cytotoxicant, and acetaldehyde, a weak clastogen. Cell death, proposed to be the result of intracellular acidification, results in restorative cell proliferation. In conjunction with sufficient genetic damage, induced by spontaneous mutation and acetaldehyde-induced DNA-protein cross-links (DPX), olfactory degeneration progresses to a state of elevated proliferation and eventually, at high vinyl acetate concentrations, to neoplastic transformation. Thus, reduction in intracellular pH (pHi) is proposed as the dosimeter most closely linked to the earliest stages of vinyl acetate toxicity. Consequently, risk assessments that are based on protection of nasal epithelium from intracellular acidification will be protective of all subsequent pathological responses related to vinyl acetate exposure. Proposing a reasonable mode of action is an important step in any risk assessment and is critical to the choice of dosimeter(s) to be used for interspecies dosimetry extrapolation.

Role of prolactin in chloro-S-triazine rat mammary tumorigenesis.

Chloro-S-triazine herbicides [cyanazine (CZ), atrazine (AZ), simazine (SZ)] increase mammary tumors in Crl:CD BR rats but not in F-344 rats or in mice. A nongenotoxic mechanism was investigated since the chloro-S-triazines are negative in short-term tests for genotoxicity. An in vivo battery was used to assess the chloro-S-triazines for estrogenic activity or for their ability to increase prolactin (PRL) levels, both of which play important roles in enhancing mammary gland tumorigenesis in rodents. Ovariectomized (OVX) female rats were treated with AZ, CZ, SZ, or three CZ metabolites for 4 days via intraperitoneal injection. The pattern of responses between the chloro-S-triazines and four controls (estradiol, estriol, haloperidol, reserpine) was compared. For the 6 end-points examined, the responses from rats treated with AZ, CZ, SZ, and the metabolites of CZ most closely matched the responses from the reserpine-treated rats (a PRL rather than estrogenic mechanism). In addition, AZ, CZ, and SZ were tested in several other in vitro models (estrogen/biogenic amine receptor competition assays and a yeast-expressed human estrogen receptor transcription assay) as well as an in vivo 24 h time-course experiment to characterize the CZ-induced increases in PRL levels. AZ, CZ, and SZ are not estrogen receptor (ER) activating compounds based on yeast transactivation and receptor competition data. CZ and AZ demonstrated marginal competition (at mM levels) to the D and alpha2 adrenergic receptors. Ligands to the D2 receptor, but not the alpha2 adrenergic receptor, are known to induce mammary tumors. CZ was also found to produce elevated PRL levels in a time-course similar to that seen with reserpine and haloperidol. Overall, the pattern of responses obtained with the chloro-S-triazines most closely matched the responses observed for reserpine. Taken together, these data suggest chloro-S-triazine-induced mammary tumors in rats are mediated through a PRL mechanism, which is thought to be of low relevance to humans.

A biologically based risk assessment for vinyl acetate-induced cancer and noncancer inhalation toxicity.

The 1990 Clean Air Act Amendments require that health risk from exposure to vinyl acetate be assessed. Vinyl acetate is a nasal carcinogen in rats, but not mice, and induces olfactory degeneration in both species. A biologically based approach to extrapolating risks of inhalation exposure from rats to humans was developed, which incorporates critical determinants of interspecies dosimetry. A physiologically based pharmacokinetic (PBPK) model describing uptake and metabolism of vinyl acetate in rat nose was validated against nasal deposition data collected at three airflow rates. The model was also validated against observations of metabolically derived acetaldehyde. Modifying the rat nose model to reflect human anatomy created a PBPK model of the human nose. Metabolic constants from both rats and humans specific for vinyl acetate and acetaldehyde metabolism enabled predictions of various olfactory tissue dosimeters related to the mode of action. Model predictions of these dosimeters in rats corresponded well with observations of vinyl acetate toxicity. Intracellular pH (pHi) of olfactory epithelial cells was predicted to drop significantly at airborne exposure concentrations above the NOAEL of 50 ppm. Benchmark dose methods were used to estimate the ED10 and LED10 for olfactory degeneration, the precursor lesion thought to drive cellular proliferation and eventually tumor development at excess cellular acetaldehyde levels. A concentration x time adjustment was applied to the benchmark dose values. Human-equivalent concentrations were calculated by using the human PBPK model to predict concentrations that yield similar cellular levels of acetic acid, acetaldehyde, and pHi. After the application of appropriate uncertainty factors, an ambient air value of 0.4 to 1.0 ppm was derived. The biologically based approach supports a workplace standard of 10 ppm.

Analysis of vinyl acetate metabolism in rat and human nasal tissues by an in vitro gas uptake technique.

Physiologically based pharmacokinetic (PBPK) models require estimates of catalytic rate constants controlling the metabolism of xenobiotics. Usually, these constants are derived from whole tissue homogenates wherein cellular architecture and enzyme compartmentation are destroyed. Since the nasal cavity epithelium is composed of a heterogeneous cell population measurement of xenobiotic metabolizing enzymes using homogenates could yield artifactual results. In this article a method for measuring rates of metabolism of vinyl acetate, a metabolism-dependent carcinogen, is presented that uses whole-tissue samples and PBPK modeling techniques to estimate metabolic kinetic parameters in tissue compartments. The kinetic parameter estimates were compared to those derived from homogenate experiments using two methods of tissue normalization. When the in vitro gas uptake constants were compared to homogenate-derived values, using a normalization procedure that does not account for tissue architecture, there was poor agreement. Homogenate-derived values from rat nasal tissue were 3- to 23-fold higher than those derived using the in vitro gas uptake method. When the normalization procedure for the rat homogenate-derived values took into account tissue architecture, a good agreement was observed. Carboxylesterase activity in homogenates of human nasal tissues was undetectable. Using the in vitro gas uptake technique, however, carboxylesterase activity was detected. Rat respiratory carboxylesterase and aldehyde dehydrogenase activities were about three and two times higher than those of humans, respectively. Activities of the rat olfactory enzymes were about equivalent to those of humans. K(m) values did not differ between species. The results suggest that the in vitro gas uptake technique is useful for deriving enzyme kinetic constants where effects of tissue architecture are preserved. Furthermore, the results suggest that caution should be exercised when scaling homogenate-derived values to whole-organ estimates, especially in organs of cellular heterogeneity.

Physiologically based modeling of vinyl acetate uptake, metabolism, and intracellular pH changes in the rat nasal cavity.

Chronic inhalation exposure to vinyl acetate (VA) causes lesions in the nasal cavity of the rat. This effect appears to be related to tissue exposure to either acetaldehyde (AAld) or acetic acid (AA) metabolites of VA or both. A physiologically based pharmacokinetic model was constructed to describe the deposition of VA in the nasal cavity of the rat and provide estimates of regional tissue exposure to VA, AAld, and AA. Since formation of AA in the nasal tissue should cause intracellular acidification, a submodel which describes free intracellular hydrogen ion concentration and intracellular pH (pHi) changes was linked to the VA model. The dosimetry model was applied to data from a series of experiments designed to measure the uptake and metabolism of VA in the isolated upper respiratory tract of the rat at exposure concentrations ranging from 73 to 2190 ppm. Extraction of VA from the nasal cavity was nonlinear with respect to exposure concentration and ranged from 36 to 94%, with the greatest deposition occurring at the lowest VA concentrations. Pretreatment with bis(p-nitrophenyl)phosphate, an inhibitor of carboxylesterases, significantly reduced fractional deposition of VA compared to naive rats exposed to similar VA concentrations. The best model fits for VA extraction and AAld appearance were achieved when a second carboxylesterase isozyme, with high-affinity characteristics, was included. Simulations of 6-h inhalation exposures to VA predicted that the order of nasal tissue exposures will be to AA > AAld > VA. In addition, based on measured tissue hydrolysis rates, sufficient acid should be formed by the metabolism of VA to cause significant changes in pHi. VA exposures of 200 and 600 ppm were predicted to result in a pHi of less than 7.2 and 6.7, respectively. This model provides nasal dosimetry estimates needed to develop mechanistically based risk assessment approaches for human exposures to VA vapor.

A physiologically based pharmacokinetic model for nicotine disposition in the Sprague-Dawley rat.

A physiologically based pharmacokinetic (PBPK) model was developed to describe the disposition of nicotine in the Sprague-Dawley (SD) rat. Parameters for the model were either obtained from the literature (blood flows, organ volumes) or determined experimentally (partition coefficients). Nicotine metabolism was defined in the liver compartment by the first-order rate constants KNC and KNP which control the rate of nicotine metabolism to cotinine and "polar metabolites" (PM), respectively. These rate constants were estimated by optimizing the model fit to pharmacokinetic data obtained by administering an intraarterial (S)-[5-3H]nicotine bolus of 0.1 mg/kg to 6 rats. Model simulations that optimized for the appearance of cotinine in plasma estimated KNC and KNP to be 75.8 and 24.3 hr-1, respectively. Use of these constants in the model allowed us to accurately predict nicotine plasma kinetics and the fraction of the dose eliminated by renal (8.5%) and metabolic (91.5%) clearance. To validate the model's ability to predict tissue kinetics of nicotine, 21 male SD rats were administered 0.1 mg/kg (S)-[5-3H]nicotine intraarterially. At seven time points following treatment, 3 rats were euthanized and tissues were removed and analyzed for nicotine. Model-predicted nicotine tissue kinetics were in agreement with those determined experimentally in muscle, liver, skin, fat, and kidney. The brain, heart, and lung exhibited nonlinear nicotine elimination, suggesting that saturable nicotinic binding sites may be important in nicotine disposition in these organs. Inclusion of saturable receptor binding expressions in the mathematical description of these compartments resulted in better agreement with the experimental data. The Bmax and KD estimated by model simulations for these tissues were brain, 0.009 and 0.12; lung, 0.039 and 2.0; and heart, 0.039 nmol/tissue and 0.12 nM, respectively. This PBPK model can successfully describe the tissue and plasma kinetics of nicotine in the SD rat and will be a useful tool for pharmacologic studies in humans and experimental animals that require insight into the plasma or tissue concentration-effect relationship.

The effect of benzo(a)pyrene on murine ovarian and corpora lutea volumes.

OBJECTIVE: Women who smoke have impaired fertility and experience menopause at an earlier age. This experiment determined the effect of benzo(a)pyrene, a polycyclic aromatic hydrocarbon contained in cigarette smoke, on murine ovarian volume, total corpora lutea volume, individual corpora lutea volumes, and corpora lutea numbers. STUDY DESIGN: C57BL/6N mice were treated with intraperitoneal injections of 0 to 500 mg/kg benzo(a)pyrene in corn oil. The 20 mice at each dose were divided into four groups of five each and were killed at 1, 2, 3, or 4 weeks after treatment. Ovaries were serially sectioned and analyzed morphometrically. RESULTS: Benzo(a)pyrene produced a dose- and time-dependent decrease in ovarian volume, total corpora lutea volume, and number of corpora lutea per ovary. This effect was transitory at low doses with complete recovery of corpora lutea by 4 weeks. Compensatory hypertrophy of the individual corpora lutea occurred during the recovery phase. Ovarian function did not return in animals treated with the two highest doses. CONCLUSION: Benzo(a)pyrene is a murine ovarian toxicant that inhibits corpus luteum formation in a dose- and time-dependent fashion.

Reproductive toxicity of cyclophosphamide in the C57BL/6N mouse: 1. Effects on ovarian structure and function.

The antineoplastic alkylating agent, cyclophosphamide (CPA) is known to impair normal female reproductive function. We have examined the time- and dose-dependent effects of CPA on the ovary, specifically, its impact on follicle numbers, ovarian morphometrics, and estradiol (E2) production. Female C57BL/6N mice were treated ip with CPA in normal saline at doses of 0, 75, 200, or 500 mg/kg. Ovaries were removed 1 to 14 days following treatment and serial sections were prepared. Differential follicle counts revealed that primordial follicles were most sensitive to CPA (ED50 = 122 mg/kg), followed by antral and growing follicles. Primordial follicles were affected by all doses of CPA and were completely destroyed by 3 days in the 500 mg/kg dose group. The greatest reduction in antral follicles was to 49% and 7% of controls by CPA doses of 200 and 500 mg/kg, respectively. Plasma E2 concentrations correlated best with antral follicle numbers (r2 = 0.94) and antral follicle volume (r2 = 0.88). Growing follicles were least sensitive to CPA and only decreased at 7 and 14 days. Although atretic changes were observed in growing follicles after treatment with CPA, these follicles recovered and progressed into apparently functional antral follicles (that is, they produced E2). Total ovarian volume was significantly reduced (30 to 40%) in the high-dose group on day 1, and remained depressed throughout the experiment. Examination of ovarian morphometrics indicated that this volume loss represented specific temporal changes in corpora lutea (CL), interstitial tissue, growing follicles, and antral follicles. At 1 and 3 days after treatment, the major loss in ovarian volume was due to a reduction in antral follicle and interstitial tissue volumes, while at 7 days the majority of volume loss was accounted for by the absence of CL. It is not known if CL are directly affected by CPA at the early time points, but their absence at 7 and 14 days is probably due to earlier destruction of antral follicles. These results demonstrate that CPA-induced ovarian toxicity is exhibited as temporal changes in both structural and functional features of the ovary, particularly in destruction of primordial and antral follicles and depressed E2 production. Information of this type also gives insight into ovarian response to chemical disruption of folliculogenesis and its recovery process.

Reproductive toxicity of cyclophosphamide in the C57BL/6N mouse: 2. Effects on uterine structure and function.

Cyclophosphamide-induced uterine weight loss was evaluated to determine whether it was a function of primary toxicity to the uterus or a secondary response to ovarian toxicity, that is, antral follicle destruction. C57BL/6N mice treated with cyclophosphamide exhibited a reduction in uterine weight concurrent with a decrease in plasma estradiol (E2) concentrations, thereby indicating toxicity to the ovary. However, when E2 concentrations recovered, uterine weight still remained depressed, suggesting that cyclophosphamide also impaired uterine function. Further investigation revealed that cyclophosphamide altered the normal uterotropic response to E2, significantly diminishing the uterine weight gain associated with E2 treatment. We conclude that effects of cyclophosphamide on the uterus involve two components: 1) decreased uterine weight in response to decreased plasma E2 resulting from ovarian toxicity, and 2) an altered response to E2 due to direct uterine toxicity.

Phosphoramide mustard is responsible for the ovarian toxicity of cyclophosphamide.

Although cyclophosphamide (CPA) is an ovarian toxicant, the responsible metabolite(s) have not been identified. The purpose of these experiments was to determine if phosphoramide mustard or acrolein were the proximate toxicants produced by metabolic activation of CPA. To do this analogs of CPA known to generate either phosphoramide mustard or acrolein in vivo were assessed for their ability to produce ovarian toxicity as measured by differential follicle destruction, ovarian volume loss, and uterine weight loss and compared to the effects produced by CPA. Phosphoramide mustard cyclohexylamine salt (PMC) and trans-4-phenylcyclophosphamide (T4P), both of which generate phosphoramide mustard, and didechlorocyclophosphamide (DCPA) and allyl alcohol (AA) which generate acrolein were administered ip to female C57BL/6N mice, 10-12 weeks old, at doses equimolar to 0, 25, 75, 200, or 500 mg/kg of CPA. Three days later the animals were killed, their uterine weights measured and their ovaries removed, fixed, and serially sectioned. Only PMC and T4P produced ovarian toxicity. On an equimolar basis these compounds were over twice as potent as CPA. Both caused a significant reduction in uterine weight (to 50% of controls) at doses of 200 (PMC) and 150 mg/kg (T4P). PMC and T4P also caused a 50% reduction in ovarian volume at doses above 75 mg/kg. Primordial follicles were most sensitive; ED50s were 76.9, 25.3, and 19.3 mg/kg (0.276, 0.091, and 0.069 mmol/kg) for CPA, PMC, and T4P, respectively. Growing follicle numbers were also reduced by T4P and PMC, an effect not seen with CPA treatment. Finally, antral follicles were significantly reduced by all doses of PMC, and with T4P at doses greater than 75 mg/kg. The highest doses of PMC, T4P, and CPA all caused a reduction in antral follicle numbers to less than one percent of controls. Didechlorocyclophosphamide (DCPA) and allyl alcohol (AA), compounds that generate acrolein but not phosphoramide mustard in vivo, had no effect on any of the parameters measured even when injected directly into the ovary. This suggests that phosphoramide mustard is responsible for CPA ovarian toxicity. The greater potency of PMC and T4P compared to CPA is likely the result of these compounds bypassing important detoxification steps, therefore, more of the parent compound reaches the ovary as the toxic metabolite.

Comparison of random and serial sections in assessment of ovarian toxicity.

Assessment of ovarian toxicity by follicle quantitation and morphometric analysis of serial sections is time consuming and expensive. This report compares the estimation of follicle number obtained from counting oocytes in serial sections or 5 random sections of mouse ovaries. Ovaries were obtained from C57BL/6N and B6C3F1 mice treated with ovarian toxicants. C57BL/6N mice were treated with cyclophosphamide (0, 75, 200, and 500 mg/kg, ip) and killed at 24, 72, and 168 h. B6C3F1 mice were treated daily, ip, with 4-vinylcyclohexene (0, 100, 400, and 800 mg/kg/day for 30 days), vinylcyclohexene diepoxide (0, 10, 40, and 80 mg/kg/day for 30 days), or benzo(a)pyrene (100 mg/kg, single dose) and killed on day 31. Ovarian serial sections were prepared and oocytes counted in every tenth section. When serial sections were evaluated, 30 to 60 sections were counted. Random section counting involved randomly selecting 5 sections from the 30 to 60 sections previously counted by the serial method. Chemically-induced follicle loss was evident by the reduction in follicle counts relative to control animals when estimating follicle number using serial or random section counting. Furthermore, a linear regression analysis of follicle counts over all treatment groups showed that the highest correlation between random and serial section counting and was for primordial follicles. Correlation coefficients (R2) for each follicle type were: primordial--C57BL/6N 0.82, B6C3F1 0.86; growing--C57BL/6N 0.16, B6C3F1 0.46; antral--C57BL/6N 0.07, B6C3F1 0.11. Although other screens may need to be developed for growing and antral follicles, primordial follicle toxicity is adequately defined by counting 5 random sections.

Reproductive toxicity: male and female reproductive systems as targets for chemical injury.

On the basis of current knowledge of reproductive biology and toxicology, it is apparent that chemicals affecting reproduction may elicit their effects at a number of sites in both the male and the female reproductive system. This multiplicity of targets is attributable to the dynamic nature of the reproductive system, in which the hypothalamic-pituitary-gonadal axis is controlled by precise positive and negative feedback mechanisms among its components. Interference by a xenobiotic at any level in either the male or the female reproductive system may ultimately impair hypothalamic or pituitary function. Normal gonadal processes such as spermatogenesis or oogenesis, ejaculation or ovulation, hormone production by Leydig or granulosa cells, and the structure or function of the accessory reproductive structures (e.g., epididymis, fallopian tube) also appear vulnerable to xenobiotics. The reproductive system is a complex one that requires local and circulating hormones for control. This brief review illustrates a system for characterizing the mechanism of action of reproductive toxicants, as well as for defining the sites available for disruption of reproduction. Unfortunately, at present, data addressing the actual vulnerability of reproduction are sorely lacking. However, when experiments have been conducted and combined with epidemiologic data or clinical observation, it has been possible to demonstrate impairment of reproductive processes by xenobiotics. The role of environmental exposure to xenobiotics in the increase in infertility that has been observed remains to be defined.

Effects of manganese (Mn++) and iron (Fe+++) on magnetic resonance imaging (MRI) characteristics of human placenta and amniotic fluid.

The contrast and intensity of a magnetic resonance image (MRI) is affected in part by the spin-lattice relaxation time (T1) and spin-spin relaxation time (T2). Certain paramagnetic metal ions can alter these parameters suggesting that they may be useful contrast agents in MRI. In this study, Mn++ and Fe+++ were examined for their effects on T1 and T2 in human placenta and amniotic fluid (AF) at concentrations between 0.002 and 2.0 mM. Both Mn++ and Fe+++ produced a dose-dependent decrease in placental and AF T1. The effects of Fe+++ were not pronounced, decreasing T1 only at the highest concentrations, and not to the same degree as Mn++. Placental T2 was also significantly decreased by Mn++, whereas Fe+++ had no effect. These differences may be due to molecular binding, uptake by the placenta, or the paramagnetic characteristics of the metals. The results suggest that Mn++ will alter human placental MRI for T1 and to a lesser extent T2-dependent imaging processes. Fe+++ should have little or no effect on human placental MRI, except at very high concentrations.