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.

Toward a biologically based dose-response model for developmental toxicity of 5-fluorouracil in the rat: a mathematical construct.

Biologically based dose-response (BBDR) models comprise one way to incorporate mechanistic information into a dose-response assessment to be used for risk assessments. The chemotherapeutic drug 5-fluorouracil (5-FU) has been used as a prototypic compound for the construction of a BBDR model for developmental toxicity. Previous work has provided data and a general mechanistic framework for the developmental toxicity of 5-FU when it was administered to pregnant rats subcutaneously on gestation day 14. In this paper, a mathematical model relating maternally administered treatment with 5-FU to embryonal thymidylate synthetase inhibition and thymidylate synthetase inhibition to various measures of deoxyribonucleotide triphosphate (dNTP) pool perturbation is developed, and parameters are estimated using the data collected. The strategy used was to develop semi-empirical submodels for each of the intervening steps, and to estimate model parameters from previously described data. The models developed predict that there is no practical threshold for dNTP pool perturbation; that is, even minimal doses of 5-FU should result in some perturbation of dNTP pools. In particular, the relationship between dNTP pool perturbation and fetal weight deficit suggests that if there is a biological threshold for the effect of 5-FU on fetal weight, the responsible repair or compensation mechanism must be downstream of dNTP pool perturbation, and saturable at 5-FU doses lower than 10 mg/kg (the lowest dose examined for developmental effects in these studies).

Comparison of in vivo and in vitro methods for assessing the effects of repeated dosing with carbon tetrachloride on the hepatic drug-metabolizing enzyme system.

The effect of 7 daily i.p. injections of 0, 2, 20, or 200 microliters/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay, employing lindane (gamma-hexachlorocyclohexane), and by a battery of in vitro enzyme assays. The data in this study indicated that repeated administration of CCl4 for 7 days significantly increased phase I and phase II reactions in vivo and in vitro. Though there were differences between the responses of the in vivo and in vitro assays, this is the first report of increased hepatic drug-metabolizing enzyme activity from repeated treatment with CCl4.

Comparison of in vivo and in vitro methods for assessing the effects of carbon tetrachloride on the hepatic drug-metabolizing enzyme system.

The effect of a single i.p. injection of 0, 20, 200, and 1000 microliter/kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that carbon tetrachloride had a biphasic influence on the phase I reactions with the lowest dose inducing a significant increase in enzyme activity while the highest dose produced significant inhibition. Significant CCl4-induced reductions in glucuronyltransferase and sulfotransferase activities were also observed while the effect on glutathione-S-transferase was ambiguous. The in vivo and in vitro assays showed good agreement.

Comparison of in vivo and in vitro methods for assessing the effects of bromobenzene on the hepatic-metabolizing enzyme system.

The effect of daily i.p. injections of 0, 0.05, 0.5 and 5.0 mmol/kg bromobenzene for 1 week on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that repeated pretreatment with bromobenzene for 1 week stimulated a dose-related increase in phase I reactions while inducing phase II reactions at the high dose (5 mmol/kg bromobenzene). The in vivo and in vitro assays showed good agreement.

Saturation of lindane metabolism in chronically treated (YS x VY) F1 hybrid mice.

The organochlorine insecticide lindane (gamma-hexachlorocyclohexane) induces hepatomas in select strains of mice including two of three phenotypic classes of (YS X VY) F1 hybrid mice. In contrast, lindane does not induce hepatomas in rats and other strains of mice. It has been suggested that variations in the biotransformation of lindane may play a role in the different susceptibility of rodents to lindane-induced hepatomas. This study reports the effect of chronic treatment with 160 ppm dietary lindane on the comparative metabolism and disposition of this insecticide in obese yellow Avy/a, lean pseudoagouti Avy/a, and lean black a/a phenotypes of (YS X VY) F1 hybrid female mice at 17, 30, 56, and 86 wk of age. At 24 h prior to necropsy, all mice were dosed po with 18 mg lindane (containing 55 muCi [U-14C]lindane)/kg. Urine, feces, and expired air were sampled for analysis. Data indicated that metabolism of lindane and excretion of its metabolites by these mice differ significantly from those of rats that are resistant to lindane-induced hepatomas. Treatment of the mice with 160 ppm lindane in the diet appeared to saturate the elimination pathways and resulted in an increased tissue burden of the insecticide and its metabolites in the older animals. Results indicate that differences in lindane metabolism and disposition observed in the (YS X VY) F1 hybrid mice were associated with chronic lindane treatment, aging, and obesity but not with genotype.

A comparison of in vitro and in vivo methods for evaluating alterations in hepatic drug metabolism following mercuric chloride administration.

Mercuric chloride was administered once i.p. to female Fischer-344 rats at doses of 0, 0.2, 0.6 and 1.8 mg/kg. Although there were no alterations in the urinary excretion of lactate dehydrogenase, significant elevations in the activities of urinary (U) alkaline phosphatase, glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) indicated that mercuric chloride was nephrotoxic. There was no evidence of hepatotoxicity as hepatic glucose-6-phosphatase and serum sorbitol dehydrogenase were essentially unaffected by mercuric chloride administration. The activities of ethylmorphine demethylase, hexobarbital oxidase and aldrin epoxidase determined in vitro were not inhibited by mercuric chloride although aniline hydroxylase activity was decreased. Of the four phase-II reactions measured, only the glucuronidation of chloramphenicol was diminished by treatment with mercuric chloride. Results from the in vivo studies on the metabolism of lindane, which indicated no change in the excretion of free or conjugated metabolites, were in close agreement with the in vitro data suggesting that the nephrotoxic effects of mercuric chloride do not alter the urinary excretion of the model substrate lindane.

Use of gamma-hexachlorocyclohexane (lindane) to determine the ontogeny of metabolism in the developing rat.

The compound lindane (gamma-hexachlorocyclohexane) has been used to study the ontogeny of metabolism in the developing Fischer 344 rat. The distribution and metabolic fate of lindane at 2, 9, 16, and 23 d of age was investigated following subcutaneous administration of lindane at 20 mg/kg containing 0.5 microCi [U-14C]lindane in peanut oil. Groups of 10 pups (5 male and 5 female) were sacrificed at 4-h intervals during the 24-h period following dosing. Adrenals, blood, brain, heart, lung, liver, and kidneys were analyzed for radioactivity. Urine samples were analyzed for radioactivity and metabolites of lindane. There was a significant age-dependent increase in the metabolism of lindane in the rat. High levels of radioactivity in the lung and increased reductive dechlorination suggest that the lung may play a greater role in metabolism of lindane by young rats. Oxidative phase I reactions increased significantly, while anaerobic reductive dechlorination of lindane to 4-chlorophenylmercapturic acid decreased significantly with age. Phase II sulfate and glutathione conjugations decreased significantly and glucuronide conjugation increased significantly with age. Metabolism and excretion of lindane appear to parallel development of the hepatic enzymes involved in phase I and phase II reactions.

Comparison of in vivo and in vitro methods for assessing effects of allyl alcohol on the liver.

Allyl alcohol was administered intraperitoneally (i.p.) to female Fischer 344 rats at doses of 0, 3, 10 and 30 mg/kg daily for 7 days. Plasma sorbitol dehydrogenase was minimally elevated. No dose-related changes were observed in hexobarbital oxidation, aniline hydroxylation, or ethylmorphine demethylation. Aldrin epoxidation was slightly elevated. Naphthol glucuronidation and glutathione-S-transferase activity with 1,2-dichloro-4-nitrobenzene were increased. Results from in vivo studies on the metabolism of lindane were in close agreement with the in vitro measurements suggesting that daily treatment for one week with allyl alcohol at doses of 3, 10 and 30 mg/kg has no significant effect on phase I pathways, has a selective effect on phase II pathways and, under the conditions of this experiment, has minimal hepatotoxic effects in these rats.

Comparison of in vitro methods and the in vivo metabolism of lindane for assessing the effects of repeated administration of ethanol on hepatic drug metabolism.

In vitro methods of assessing alterations in drug metabolism and the measurement of lindane metabolites in urine were compared for their ability to determine the influence of ethanol on drug metabolism. Ethanol was administered intraperitoneally (i.p.) to young adult female rats daily for 7 days at doses of 0.12, 0.60 and 3.0 ml/kg. No alterations were observed in ethylmorphine demethylation, hexobarbital oxidation or glucuronyltransferase. Aniline hydroxylation was decreased at the high dose level and aldrin epoxidation was increased at the intermediate dose. In vivo only the high dose of ethanol produced significant changes with significant increases observed for the oxidation of lindane to alcohol metabolites, the glucuronidation of the alcohol but not the chlorophenol metabolites, and glutathione conjugation. The latter increase was also observed in vitro. The in vivo and in vitro data suggest a minimal effect of ethanol on drug metabolism at low levels of administration.

Comparison of in vivo and in vitro methods for assessing the effects of phenobarbital on the hepatic drug-metabolizing enzyme system.

The effect of daily i.p. injections of 0, 1, 10 and 80 mg/kg phenobarbital for 1 week on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (gamma-HCH) and by a battery of in vitro enzyme assays. Comparison of the dose-response curves of the in vivo and in vitro assays indicated that urinary metabolites of lindane provided a good index of phenobarbital-induced change in both phase I and phase II reactions.

Investigation of HCB as a metabolite from female rats treated daily for six days with lindane.

The biotransformation of lindane to hexachlorobenzene (HCB) by male rats was recently reported. Since HCB has been widely detected in human milk samples, and since the transplacental transfer of HCB to the fetus has been demonstrated in several species, the metabolism of lindane to HCB in female rats was investigated. Young adult female Fischer 344 rats were dosed p.o. with either 20 mg lindane/kg/day or an equivalent volume of the peanut oil vehicle. Feces samples were collected daily for two consecutive 4-hr intervals and a 16-hr interval. Twenty-four hours after the final treatment, all rats were sacrificed and adipose tissue samples were excised at necropsy. Extracts of fat and feces samples were analyzed by gas-liquid chromatography (GLC) on column packings of different polarity. Results of this study indicated that no significant biotransformation of lindane to HCB occurred in the female Fischer 344 rat.

Effects of age and obesity on the metabolism of lindane by black a/a, yellow Avy/a, and pseudoagouti Avy/a phenotypes of (YS x VY) F1 hybrid mice.

Lindane (gamma-hexachlorocyclohexane) has been shown to produce hepatomas in some strains of mice but not in others. Genetic factors and/or altered metabolism may play a role in the susceptibility to lindane-induced hepatomas. This study reports the effect of age and obesity on the comparative metabolism and disposition of lindane in obese yellow Avy/a and in lean pseudoagouti Avy/a and black a/a phenotypes of (YS x VY) F1 hybrid female mice at 8, 17, 30, 56, and 86 wk of age. At 24 h prior to sacrifice the mice were dosed p.o. with 18 mg lindane (containing 55 microCi [U-14C]lindane/kg). Aging altered the biotransformation of lindane such that while the excretion of lindane and its metabolites declined, the proportion of conjugated and polar metabolites increased. Tissue storage was elevated in older animals. In the yellow Avy/a mice, which are known to have a predisposition to the formation of hepatomas, there was accelerated and prolonged growth, reduced metabolite excretion, a greater proportion of conjugated metabolites, and higher dechlorinase activity compared to that of their pseudoagouti Avy/a and black a/a siblings.

Chlorobenzene-impaired lindane metabolism and the effect of pretreatment with chlorobenzene, lindane, or chlorobenzene plus lindane.

The storage and metabolism of lindane (gamma-HCH) was studied in the female rat after the administration of a hepatotoxic dose of chlorobenzene. Impaired lindane metabolism was observed following a challenge dose of 1.12 g chlorobenzene/kg. The data indicated that a hepatotoxic dose of chlorobenzene (CB) selectively impaired certain pathways, such as dehydrochlorination and the direct hydroxylation of lindane, to a greater extent than others, such as the dehydrogenation and dechlorination of lindane. Pretreatment with a subtoxic level of chlorobenzene produced: (1) significant increases in the dehydrogenation of lindane, (2) significant increase in the excretion of conjugated metabolites, (3) significant increases in the excretion of metabolites derived from the dehydrogenation of lindane through hexachlorocylohexene, gamma-HCCH, (4) significant improvement in the excretion of metabolites derived from CB-impaired dehydrochlorination of lindane as well as from the CB-impaired hydroxylation of lindane, and (5) significant reduction in the level of unaltered lindane stored in the adipose tissue. Repeated pretreatment with a subtoxic level of chlorobenzene offered significant protection against the reduction in lindane metabolism produced by the single hepatotoxic dose of chlorobenzene. Pretreatment with gamma-HCH alone was not as effective against the hepatotoxic effect of CB on lindane metabolism.

Bioisomerization of lindane in rats.

The major environmental problem associated with the use of gamma-hexachlorocyclohexane (gamma-HCH, lindane) has been the appearance of the more oncogenic alpha- and beta- isomers as terminal residues in nature. To account for these residues it was suggested that gamma-hexachlorocyclohexane had been bioisomerized to the more stable alpha- and beta- isomers. In this study the effect of dose and duration of treatment on the proposed bioisomerization of gamma-hexachlorocyclohexane in the rat was investigated. Weanling female Sprague-Dawley rats were randomly assigned to one of four groups receiving Purina Lab Chow fortified with 0, 130, 215, or 350 ppm gamma-HCH. Six animals from each group were sacrificed after 1, 2, 4, 8, 16, and 24 weeks of treatment. Twenty-four hours prior to sacrifice all rats received a single oral dose of gamma-HCH in peanut oil. There were no significant differences in food consumption or body weights, and no deaths occurred throughout the study. The in vitro dechlorinase activity of the treated rats was significantly higher after 1, 4, and 24 weeks of treatment. Except at 4 weeks after treatment began, the liver/body weight ratios of the rats fed diets containing 350 ppm and 215 ppm lindane were significantly greater than the controls; while those receiving 130 ppm lindane were significantly greater than the controls after 1 and 2 weeks of treatment. No beta-HCH was detected in any of the samples analyzed throughout the study. The levels of alpha-HCH found in the adipose tissue after 24 weeks of treatment could be accounted for by trace contamination of the lindane used in this study. There was a negative correlation between the hepatic content of alpha-, gamma-, and sigma-HCH and duration of treatment. It was concluded that bioisomerization does not play a significant role in the metabolism of lindane by rats.