Toward a biologically based dose-response model for developmental toxicity of 5-fluorouracil in the rat: acquisition of experimental data.

Biologically based dose-response (BBDR) models represent an emerging approach to improving the current practice of human health-risk assessment. The concept of BBDR modeling is to incorporate mechanistic information about a chemical that is relevant to the expression of its toxicity into descriptive mathematical terms, thereby providing a quantitative model that will enhance the ability for low-dose and cross-species extrapolation. Construction of a BBDR model for developmental toxicity is particularly complicated by the multitude of possible mechanisms. Thus, a few model assumptions were made. The current study illustrates the processes involved in selecting the relevant information for BBDR modeling, using an established developmental toxicant, 5-fluorouracil (5-FU), as a prototypic example. The primary BBDR model for 5-FU is based on inhibition of thymidylate synthetase (TS) and resultant changes in nucleotide pools, DNA synthesis, cell-cycle progression, and somatic growth. A single subcutaneous injection of 5-FU at doses ranging from 1 to 40 mg/kg was given to pregnant Sprague-Dawley rats at gestational day 14; controls received saline. 5-FU was absorbed rapidly into the maternal circulation, and AUC estimates were linear with administered doses. We found metabolites of 5-FU directly incorporated into embryonic nucleic acids, although the levels of incorporation were low and lacked correlation with administered doses. On the other hand, 5-FU produced dose-dependent inhibition of thymidylate synthetase in the whole embryo, and recovery from enzyme inhibition was also related to the administered dose. As a consequence of TS inhibition, embryonic dTTP and dGTP were markedly reduced, while dCTP was profoundly elevated, perhaps due to feedback regulation of intracellular nucleotide pools. The total contents of embryonic macromolecules (DNA and protein) were also reduced, most notably at the high doses. Correspondingly, dose-related reductions of fetal weight were seen as early as GD 15, and these deficits persisted for the remainder of gestation. These detailed dose-response parameters involved in the expression of 5-FU developmental toxicity were incorporated into mathematical terms for BBDR modeling. Such quantitative models should be instrumental to the improvement of high-to-low dose and cross-species extrapolation in health-risk assessment.

Pharmacokinetics of (R,S)-Albuterol after aerosol inhalation in healthy adult volunteers.

The pharmacokinetics of inhaled (R,S)-albuterol following pulmonary absorption were studied in healthy human subjects. Ten subjects (5 females and 5 males) inhaled two puffs (180 microg) of albuterol via a metered-dose inhaler and spacer device. All subjects were nonsmoking and had normal pulmonary function. Charcoal slurries were ingested to block gastrointestinal absorption of drug. Venous samples were obtained from each subject at thirteen time points from 0 through 12 h post dose. (R,S)-Albuterol concentration in plasma was measured using a gas chromatography-mass spectrometry (GC-MS) assay. The plasma concentration-time profiles conformed to a two-compartment extravascular model with first-order absorption kinetics. The drug levels reached maximum in 12.6 +/- 2.2 (SD) minutes, which is in contrast with previous reports that maximum plasma concentrations occur within 2 to 4 h. The mean peak plasma level was 1469 +/- 410 pg/mL. The mean half-life of distribution was 17.9 +/- 8.2 min. The mean half-life of elimination was 4.4 +/- 1.5 h. Female subjects achieved peak concentration more rapidly than male subjects (10.4 vs 14.8 min, p = 0.01) and had a higher mean peak concentration (1778 vs 1159 pg/mL, p = 0.04).

Determination of albuterol in plasma after aerosol inhalation by gas chromatography-mass spectrometry with selected-ion monitoring.

Albuterol is a beta2-adrenergic agonist commonly used as a bronchodilator for the treatment of patients with asthma. We have developed an assay to determine plasma levels as low as 50 pg/ml of albuterol by gas chromatography-mass spectrometry (GC-MS). This assay utilizes isotopically labeled albuterol ([13C]albuterol) as an internal standard. In this assay albuterol and the internal standard are recovered from 1 ml of plasma using solid-phase extraction. The samples are then derivatized to trimethylsilyl ethers using N,O-bis(trimethylsilyl)trifluoro-acetamide with 1% trimethylchlorosilane. The samples are then analyzed by GC-MS with selected-ion monitoring (SIM) for the ions m/z 369.15 and 370.15. The method has been validated for a concentration range of 50-10000 pg/ml in plasma.

Gamma-glutamyl transferase (GGT) activity and biochemical characterization of rat visceral yolk-sac during gestation with or without trypan blue exposure.

Yolk-sacs from untreated Sprague-Dawley rat conceptuses were removed on gestational days (GD) 9 to 18 and examined for gamma-glutamyl transferase (GGT), alkaline phosphatase (AP), lactate dehydrogenase (LDH), and glutamic-oxaloacetic transaminase (GOT) activities. All enzyme activities were found to increase through gestation in total activity as well as in specific activity. Protein (PRO) and urea nitrogen (UN) were also found to increase through gestation whereas triglyceride (TRI) increased steadily from GD 9 to 13 and then appeared to plateau through GD 19. Additional rats were treated on GD 8 with 75 mg trypan blue (TB)/kg body weight and yolk-sacs taken on GD 9 to 18. Yolk-sac GGT and GOT activities from TB-treated rats were significantly higher than the respective controls during early gestation but recovered to or were lower than control levels during midgestation. GGT activity in treated rats was significantly higher than the controls on GD 15 and 16, and both GGT and GOT were significantly lower than controls on GD 17 and 18. AP activity in the TB-treated yolk-sacs was significantly lower than that in controls during early and midgestation but was not significantly different from the control values late in gestation. Triglyceride concentration was not affected early in gestation but significantly decreased on GD 16 and 18. Thus, the yolk-sac enzymes monitored, which are associated with nutrition and normal growth, increased in activity through GD 18. The yolk-sac toxicant, trypan blue, significantly affected enzymatic activity at various time periods during gestation and resulted in significant changes in yolk-sac protein and triglyceride content.

Biologically based dose-response modeling in developmental toxicology: biochemical and cellular sequelae of 5-fluorouracil exposure in the developing rat.

Mechanistically based dose-response models for developmental toxicity require elucidation of critical biological events that intervene between maternal exposure and adverse developmental outcome. We have examined some of the major events in the rat embryo/fetus following a subcutaneous injection of 5-fluorouracil (5-FU; 0-40 mg/kg) to the dam on Day 14 of gestation. This treatment resulted in reduced fetal weight that was significant at doses of 20 mg/kg and higher, generalized reduced ossification at doses above 25 mg/kg, and wavy ribs at doses of 30 mg/kg and higher. Numerous malformations including cleft palate and hindlimb defects were substantially increased at doses of 35 and 40 mg/kg. 5-FU inhibits thymidylate synthetase (TS), resulting in inhibited growth of rapidly proliferating tissues. To identify early events in the pathogenesis of hindlimb defects, we examined the effects of 5-FU on TS activity, cell cycle, growth, and morphology in the developing hindlimb as a function of dose and time. The rate of decline of TS activity following 5-FU exposure was dose related, although maximal inhibition and recovery were similar at doses within (20 and 40 mg/kg) and below (10 mg/kg) the range of detectable developmental toxicity. Flow cytometric analysis of nuclei from embryonic hindlimbs revealed a transient increase in the percentage of cells in S phase and decrease in G0/G1 phase 8 hr after maternal injection of 20-40 mg 5-FU/kg, but not at lower doses. Reduction in growth and morphometric changes of hindlimbs were observed only after maternal exposure to 40 mg/kg. The tissue specificity of these effects was examined by comparing the hindlimb with other embryonic tissues. There was also a dose-related decline of TS activity in the embryonic liver. However, the pattern of recovery of TS activity and cell cycle alterations were different in the liver than in the hindlimb, probably reflecting the higher cell proliferative rate in the liver at this stage. We have derived a quantitative, empirical model for induction of hindlimb defects based on TS inhibition and subsequent cellular events following 5-FU exposure. The model predicted a dose response similar to that of the observed data although the predicted curve was shifted toward lower doses. These results suggest that while this model may not capture all of the critical events involved in the induction of hindlimb defects following maternal exposure to 5-FU, it does reflect a central mechanism of its developmental toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of methanol on embryonic mouse palate in serum-free organ culture.

Methanol has widespread applications in industry and manufacturing and is under consideration as an alternative automotive fuel. Human exposure to methanol would be expected to increase if applications expand in coming years. Methanol has been shown to be a reproductive and developmental toxicant in the rodent, producing cleft palate in the CD-1 mouse. Developmental toxicity has also been demonstrated in vitro for rat and mouse embryos in whole embryo culture. The present study examines the developmental toxicity of methanol in the palate using a serum-free organ culture model. Gestation day 12 CD-1 mouse embryos were dissected and mid-craniofacial tissues were cultured in BGJ medium at 37 degrees C for 4 days with medium changes at 24 hr intervals. Cultures were exposed to methanol from 0-20 mg/ml for 6 hr, 12 hr, 1 or 4 days. Some cultures were exposed to ethanol for 4 days at doses ranging from 0-15 mg/ml. All cultures were gassed with a 50% O2, 5% CO2, and 45% N2 upon addition of fresh medium and prior to the addition of alcohol. Following organ culture the craniofacial explants were examined for effects on morphology, fusion, proliferation, and growth. Incidence and completeness of palatal fusion decreased with increasing exposure. Depending on the concentration and duration of methanol exposure, the medial epithelium either degenerated completely or remained intact in unfused palates and either condition would interfere with fusion. Cellular proliferation appeared to be a specific and sensitive target for methanol as craniofacial tissues responded to methanol with reduction in total DNA content at an exposure that did not affect total protein. However, both DNA and protein decreased with increasing exposure to methanol. Incorporation of thymidine decreased significantly after 4 day exposure and autoradiography of 3H-thymidine (TdR) demonstrated exposure-dependent reduction in proliferation of palatal mesenchymal cells. Ethanol decreased fusion score, total protein, and DNA, but 3H-TdR/DNA was not significantly changed. In general the ethanol was more potent than methanol for inhibition of protein and DNA synthesis and palatal fusion. This study demonstrated that methanol can selectively affect specific sensitive cell populations and has effects on proliferation and cell fate.

Fetal development in the rat following disruption of maternal renal function during pregnancy.

Pregnant Sprague-Dawley rats received subcutaneous injections of mercuric chloride (1-4 mg/kg) on either gestation day 7, 9, 11, or 13 to determine effects of altered maternal renal function on embryonic and fetal development. Maternal renal function, assessed by urinalysis, was markedly disrupted for at least 48 hours after treatment and resulted in decreased maternal body weight gain. Residual effects on maternal kidney weight were evident on GD 21 when the females were killed and the fetuses removed and examined for visceral and skeletal development. We did not observe an increased incidence of malformations in the offspring for exposure on any day of gestation. Maternal exposure to mercuric chloride slightly impaired fetal growth over several gestational exposures periods and changed the pattern of rib formation when exposure occurred early in organogenesis. The extent of the changes could not, however, be related to the immediate degree or duration of altered maternal renal function. Rather, we found correlations between lasting effects of exposure as measured by maternal renal weight on GD 21 and super-numerary lumbar rib induction on GD 7 and 9; while for the relationship with fetal weight, the strongest correlation with maternal kidney weight occurred following exposure on GD 9 (P < 0.01), with weaker correlations (P < 0.10) for GD 7 and 13 exposures. Maternal serum urea was negatively correlated with fetal weight from the GD 7 exposure. This study supports the concept that some specific forms of maternal toxicity may be associated with limited manifestations of developmental toxicity, but, in general, embryonic development was observed to proceed normally in the presence of marked disruptions in maternal renal physiology. Maternal toxicity, especially as routinely measured in developmental toxicity studies, cannot be looked upon as a single disease. Instead, each manifestation must be examined for what it signifies to the physiology and well being of the female and for cause-and-effect relationships with fetal observations. Maternal toxicity and developmental toxicity should not be etiologically linked solely because of their concurrent appearance on the dose-response curve.

Developmental toxicity of methanol in whole embryo culture: a comparative study with mouse and rat embryos.

Methanol (MeOH), a widely used industrial solvent, has been proposed as an alternative motor vehicle fuel. Inhaled MeOH is developmentally toxic in both rats and mice but the mouse is more sensitive than is the rat. The contribution of the embryo to this differential sensitivity was studied in whole embryo culture (WEC) using equivalent stage rat (day 9) and mouse (day 8) embryos (plug day = day 0). Rat embryos were explanted and cultured in 0, 2, 4, 8, 12 or 16 mg MeOH/ml rat serum for 24 h and then transferred to rat serum alone for 24 h. Embryonic development of the 2 and 4 mg MeOH/ml groups was not significantly different from the controls whereas the higher concentrations resulted in a concentration related decrease in somite number, head length and developmental score. The 12 mg/ml dose resulted in some embryolethality as well as dysmorphogenesis, while the highest dose was embryolethal. MeOH was dysmorphogenic in vitro in rat embryos at a MeOH concentration comparable to that reported in maternal serum following teratogenic in vivo exposures. Day 8 mouse embryos were explanted and cultured in 0, 2, 4, 6 or 8 mg MeOH/ml culture medium (75% rat serum, 25% Tyrode's salt solution) for 24 h. Embryonic development in the 2 mg/ml MeOH group was not significantly different from the controls but all higher concentration groups had a significant decrease in developmental score and crown-rump length. The high concentration group also suffered 80% embryolethality. Thus, mouse embryos were affected at MeOH concentrations which were not dysmorphogenic or embryotoxic in the rat, suggesting that the higher sensitivity of the mouse to the developmental toxicity of inhaled methanol is due, at least in part, to greater intrinsic embryonal sensitivity of this species to methanol.

Effects of 5-fluorouracil on embryonic rat palate in vitro: fusion in the absence of proliferation.

5-Fluorouracil (5-FU) inhibits the enzyme thymidylate synthetase (TS) which results in inhibition of DNA synthesis. 5-FU is teratogenic in many species, inducing cleft palate, limb, and tail defects. In the present study, gestation day (GD) 14 embryonic rat craniofacial explants were exposed to 5-FU in organ culture with increasing concentrations and durations of exposure. Palates exposed to 5-FU were morphologically abnormal and craniofacial shape, size, and palatal fusion pattern were affected with the severity of effects dependent on concentration and duration of exposure. Cleft palate was induced in vitro as opposing palates overlapped in a narrowed oral cavity. Palates exposed to higher levels of 5-FU were growth inhibited, but fused even though proliferation ceased and few cells were available to participate in elevation and fusion. This was demonstrated as a biphasic concentration-response profile for palatal fusion in which 0.05 to 0.15 micrograms 5-FU/ml produced decreasing rates of palatal fusion, while exposure to 0.15 to 3.0 micrograms/ml resulted in progressively increasing rates of fusion. The effects of 5-FU were detected biochemically as a reduction in TS activity which was concentration and time dependent during the first 12 hours, with a return to control levels by 24 hours. During the first day, 5-FU did not alter protein levels, but DNA levels significantly decreased at the high concentration, 2.0 micrograms/ml. After 5 days in culture, both DNA and protein decreased with increasing 5-FU concentration and duration of exposure. Also by the end of the culture period, 3H-TdR incorporation had decreased in a concentration dependent manner. It is concluded that progressive inhibition of proliferation and growth in organ culture results in two different morphological outcomes: cleft palate resulting from a narrowed oral cavity and increased incidence of anterior palatal fusion under conditions of strong growth reduction. This study demonstrates that elevation and fusion can occur in the absence of growth and proliferation. Based on these observations, severe inhibition of growth or proliferation would not necessarily be sufficient to induce cleft palate.

Developmental toxicity of bromohydroquinone (BHQ) and BHQ-glutathione conjugates in vivo and in whole embryo culture.

Glutathione conjugates of 2-bromohydroquinone (GSyl-BHQ) cause renal proximal tubular necrosis that is dependent upon the activity of gamma-glutamyl transferase (GGT). GGT is present in embryonic yolk sac and its activity increases with gestational age, suggesting that the developing embryo might be at risk from maternal exposure to glutathione conjugates or compounds which are shown to form glutathione conjugates. Studies in pregnant rats exposed on Day 9 of gestation to 400 or 800 mumol/kg BHQ or 20 mumol/kg 2-Br-(di-GSyl)HQ and examined on Day 11 of gestation suggested that the parent compound (BHQ) or a metabolite was nephrotoxic in the adult and dysmorphogenic in the embryo and that 2-Br-(di-GSyl)HQ was nephrotoxic but not dysmorphogenic at the dose tested (20 mumol/kg). We therefore exposed Day 9 rat embryos to BHQ, 2-Br-6-(GSyl)HQ, or 2-Br-(di-GSyl)HQ in vitro for 48 hr to determine the relative dysmorphogenic activity of the parent compounds and the two conjugates. In vitro exposure to BHQ (0-40 microM) resulted in dose-related decreases in somite number (SN), total protein, and developmental score (DEVSC), with no effect on yolk sac diameter (YSD), crown rump length (CR), head length (HL), or percentage abnormal embryos (%AE); 60 microM BHQ was embryolethal. Embryos exposed to 2-Br-6-(GSyl)HQ (0-120 microM) were not affected at concentrations below 120 microM, at which dose there were significant effects on protein, YSD, CR, HL, DEVSC, SN, and %AE. Embryos exposed to 2-Br-(di-GSyl)HQ had a significantly lower DEVSC at the 80 microM concentration and significantly lower YSD, protein, and DEVSC and significantly higher %AE at the 10, 25, and 120 microM concentrations. CR, HL, and SN were not affected at any exposure level with this compound. In conclusion, BHQ was found to be developmentally toxic in vitro and in vivo at doses which also produced severe maternal renal necrosis. The doses of 2-Br-(di-GSyl)HQ in vivo which caused only mild maternal renal necrosis did not produce developmental toxicity. Conjugation of BHQ with either one or two molecules of GSH decreased the embryolethality of BHQ. The conjugates appeared to be of comparable toxicity as measured by the incidence of abnormal embryos in vitro. The role of maternal toxicity and GSH conjugation in 2-BHQ-mediated developmental toxicity remains to be determined.

The developmental toxicity of inhaled methanol in the CD-1 mouse, with quantitative dose-response modeling for estimation of benchmark doses.

The developmental toxicity of the alternative motor vehicle fuel methanol was assessed in mice by the inhalation route. Pregnant CD-1 mice were exposed to 1,000, 2,000, 5,000, 7,500, 10,000, or 15,000 ppm methanol for 7 hr/day on days 6-15 of gestation. Sham-exposed controls were exposed to filtered air under similar conditions. Additional control groups were left in their home cages either unhandled or food-deprived for 7 hr/day to match the food deprivation experienced by the exposed mice. Dams were observed twice daily and weighed on alternate days during the exposure period. Blood methanol concentrations were determined in some mice on gestation days 6, 10, and 15. On day 17, the remaining mice were weighed and killed and the gravid uteri removed. Implantation sites, live and dead fetuses and resorptions were counted, fetuses were examined externally and weighed as a litter. Half of each litter was examined for skeletal morphology and the other half of each litter was examined for internal soft tissue anomalies. One dam died in each of the 7,500, 10,000, and 15,000 ppm methanol exposure groups, but no dose-response relationship was evident for maternal death. The sham-exposed and food-deprived controls as well as all methanol exposed dams gained less weight than did unexposed dams fed ad libitum, but methanol did not exacerbate this effect. Significant increases in the incidence of exencephaly and cleft palate were observed at 5,000 ppm and above, increased embryo/fetal death at 7,500 ppm and above (including an increasing incidence of full-litter resorptions), and reduced fetal weight at 10,000 ppm and above. A dose-related increase in cervical ribs or ossification sites lateral to the seventh cervical vertebra was significant at 2,000 ppm and above. Thus, the NOAEL for the developmental toxicity in this study was 1,000 ppm. A log-logistic dose response model was applied to the incidence data for exencephaly, cleft palate, resorption and cervical rib, and maximum likelihood estimates (MLEs) and benchmark dosages (BDs, the lower 95% confidence interval of the MLEs) corresponding to 1% and 5% added risk above background were calculated. The MLE for 5% added combined risk of having either exencephaly or cleft palate or being resorbed was 3667 ppm, and the corresponding BD was 3,078 ppm. For cervical rib, the 5% added risk values for the MLE and BD were 824 and 305 ppm, respectively. The BDs for 1% added risk were 1915 ppm for exencephaly, cleft palate or resorption, and 58 ppm for cervical rib.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro embryotoxicity of a series of para-substituted phenols: structure, activity, and correlation with in vivo data.

The embryotoxicity of phenol and twelve para-substituted congeners on mid-gestation rat embryos was evaluated in vitro. Through application of correlative procedures and stepwise regression, equations describing the relationship between physical-chemical properties and various measures of activity were developed. Embryotoxicity was quantified by the log of the reciprocal of the potency estimates for reduction in selected growth parameters and induction of four morphological defects. In general, co-cultured hepatocytes ameliorated embryotoxicity; only phenol-induced embryotoxicity was enhanced by the presence of hepatocytes. In the absence of hepatocytes, measures of growth retardation were positively correlated with molar refractivity of the phenols. With hepatocytes, lipophilicity became important for describing the potential to induce growth deficits. The structural defects had varying correlation patterns in both culture systems. Potencies of these congeners in vitro were also compared to maternal and developmental potencies observed in vivo (Kavlock, Teratology, 41:43-59, '90). Two of the congeners were very toxic in both systems. For the remaining congeners, one maternal toxicity measure was found to be positively correlated with embryotoxicity for growth and development in vitro without hepatocytes. With hepatocytes, a broad spectrum of correlations, both positive and negative, were observed between in vivo developmental toxicity endpoints and in vitro embryotoxicity. Data from preliminary dosimetry studies suggest that phenol congeners may accumulate in embryos exposed in vitro more readily than with in vivo exposure. Potency calculations based on dosimetry information may demonstrate better correlations between data and allow additional relationships between chemical structure and activity to be developed.

In vivo and in vitro structure-dosimetry-activity relationships of substituted phenols in developmental toxicity assays.

Structure-dosimetry-activity relationships (SDARs) of a series of substituted phenols were evaluated following exposure of gestation day 11 rats in vivo and in comparable stage embryos in vitro. In the in vivo study, 27 congeners were assayed and log P (a term used synomously with lipophilicity in this paper) and Hammett sigma values (a measure of the electronic withdrawing ability of the substituent) were shown to correlate with maternal toxicity; however, no relationships between these parameters and developmental effects were observed. In the in vitro system, 13 congeners were evaluated and molar refractivity and/or lipophilicity were shown to correlate with the ability of the phenols to induce embryonic growth retardation and structural defects in the absence of the hepatocytes. In contrast, when a metabolic activating system (primary hepatocytes) was present in the in vitro system, the potential to induce growth retardation was inversely related to lipophilicity, although the relationships were weaker than the positive relationship seen without the hepatocytes. The binding of the phenols to macromolecules in the culture medium was highly correlated with log P. Correcting the in vitro potency data for the variable amount of binding improved the predictiveness of the quantitative structure-activity relationships (QSARs). The potential to induce embryotoxicity in vitro was not well correlated with the potential to induce developmental toxicity in vivo: whereas the in vitro data demonstrates that the phenols are intrinsically embryotoxic, few of them actually produced significant developmental toxicity in the in vivo system, and there were few positive correlations between effects observed in the two systems.(ABSTRACT TRUNCATED AT 250 WORDS)