An evaluation of reproductive toxicity studies and data interpretation of N-methylpyrrolidone for risk assessment: An expert panel review.

An expert panel was assembled to evaluate reproductive toxicology study data and their application to health risk assessment to provide input on the data quality, interpretation, and application of data from three multi-generation reproductive toxicity studies of N-methylpyrrolidone (NMP). Panelists were engaged using a double-blinded, modified Delphi format that consisted of three rounds. Key studies were scored using the U.S. Environmental Protection Agency's (EPA) questions and general considerations to guide the evaluation of experimental animal studies for systematic review. The primary conclusions of the panel are that one of the studies (Exxon, 1991) is not a high-quality study due to several design flaws that includes: (1) exceedance of the maximum tolerable dose in the high dose group; (2) failure to adjust feed concentrations of NMP during the lactation period, resulting in NMP doses that were 2- to 3-fold higher than nominal levels; and/or (3) underlying reproductive performance problems in the strain of rats used. For these reasons, the panel recommended that this study should not be considered for quantitative risk assessment of NMP. Exclusion of this study, and its corresponding data for male fertility and female fecundity, from the quantitative risk assessment results in a change in the identification of the most sensitive endpoint. Instead, changes in rat fetal/pup body weight, an endpoint previously selected by EPA, was identified as an appropriate basis for human health risk assessment based on a consideration of the best available science and weight of scientific evidence supported by the NMP toxicity database.

Workshop to identify critical windows of exposure for children's health: cardiovascular and endocrine work group summary.

The work group on cardiovascular and endocrine effects was asked to review the current state of knowledge about children's windows of vulnerability to developmental toxicants and to recommend how that information may be used to improve risk assessment and public health. We considered differences between structural defects, where periods of vulnerability are rather well defined, and functional defects, where periods of vulnerability are quite elusive.

Geriatric bone lead metabolism in a female nonhuman primate population.

A geriatric rhesus monkey (Macaca mulatta) population, previously exposed to lead, was investigated using 109Cd K X-ray fluorescence (K XRF) to determine whether metabolism of lead in bone was similar to that in human populations. The accumulation rate of lead into the tibia in this group of monkeys was determined to be 0.10-0.13 micrograms Pb (g bone mineral)-1 (microgram dl-1 year)-1, which compares well with human data, where the rate has been found to be 0.05-0.10 microgram Pb (g bone mineral)-1 (microgram dl-1 year)-1. In addition, bone lead changes over a 10-month time period were investigated, but no statistically significant difference was found. A halflife for lead in "bone" was calculated by fitting a single exponential model to serial blood lead data; the mean half-life of lead in bone was found to be 3.0 +/- 1.0 years. Both endogenous and exogenous lead exposure were found to be low at the present time, 10 years after cessation of lead intake. It is concluded that rhesus monkeys are an extremely good animal model of human bone lead metabolism and, in addition, that further research is needed to provide a more complete understanding of lead metabolism in geriatric populations.

Chemical contaminants in human milk: an overview.

This review contains a succinct overview of the nature and extent of the problem of contamination of human milk with environmental and occupational chemicals, excluding drugs. Factors influencing the levels of contaminants in breast milk are discussed. Also, data on major chemicals of concern with potential health risk(s) to the general population and risk-benefit considerations are dealt with briefly. Based on the available data on the subject, research needs have been identified and policy recommendations are suggested.

High dietary fat feeding during perinatal development of rats alters hepatic drug metabolism of progeny.

The objective of this study was to determine whether feeding high dietary fat, during pregnancy and lactation of Sprague-Dawley rats, can modulate hepatic drug-metabolizing enzymes of the offspring during postnatal development. Time-pregnant rats were pair-fed isocalorically 20% (experimental) or 5% (control) corn oil diets from day 10 of gestation until weaning. After weaning, litters from both groups were fed 5% corn oil diet until sacrificed. Offsprings were sacrificed at weaning (23 days), puberty (45 days) and at adult stage (100 days). Feeding diet containing 20% corn oil to dams, resulted in significant increases in liver microsomal cytochrome P-450, b5 contents and aminopyrine N-demethylase activity of the male offspring at weaning, puberty and adult stage of life. A similar but less marked trend was also observed in the female offspring. Thus, it appears that the high dietary fat exposure during perinatal development may result in significant alterations in hepatic drug-metabolizing enzyme activities of the progeny.

Effect of early nutrition on serum cholesterol levels in adult rats challenged with high fat diet.

Early exposure to cholesterol-enriched, high fat diets has been reported to affect serum cholesterol levels in adult rats. In this study, we investigated the role of dietary fat alone, without cholesterol, by feeding to pregnant rats (from day 18 of gestation) experimental diets containing either high fat (corn oil), low sucrose (HF) or low fat, high sucrose (HS). After birth, mothers and pups were fed the diets until weaning (30 days), when serum cholesterol levels were the same in both groups of pups. Animals were fed stock diet until 7 months. At that time, half the animals from each original group (HF or HS) were challenged with HF diet for 3 days; the other half were fed stock diet. There was no significant difference in serum cholesterol between HF and HS animals fed stock diet: HF, 73 +/- 7 mg/dl (n = 11); HS, 80 +/- 19 (6); P greater than 0.25. Animals originally fed HF diet significantly raised serum cholesterol in response to late HF challenge (140 +/- 31 mg/dl, n = 10, P less than 0.001 vs. stock fed); the original HS-fed group did not (93 +/- 7 mg/dl, n = 5, P greater than 0.19). Early exposure to HF diet, even without cholesterol, could evoke a hypercholesterolemic response in adulthood following challenge by brief exposure to HF diet.

Effect of protein depletion and intrauterine growth retardation on rat hepatic drug metabolism.

The effect of intrauterine malnutrition in rats on in vivo and in vitro drug metabolism at weaning was investigated. We have employed two experimental designs to produce intrauterine malnutrition, maternal dietary protein depletion starting at day 7 of gestation and unilateral ligation of the uterine artery on day 17 of gestation. At birth cross-fostering of newborns was done, experimental and control group of litters raised separately until weaning (21 days of age). Both methods produced offspring with lower body and liver weights and these changes were present at weaning. However, only in the protein-restricted group were significant differences for liver:body weight ratio and hepatic microsomal protein content observed. Liver microsomal cytochrome P-450 and b5 contents were significantly decreased in small animals from mothers with intrauterine vessels ligated but not in the prenatal protein-restricted group. A significant increase in aminopyrine N-demethylase and a decrease in aniline hydroxylase enzyme activities was observed in both experimental groups. No significant differences were found in reductive or conjugative pathways of drug metabolism. Hexobarbital sleeping time was significantly increased in weanling animals from the prenatal protein-restricted mothers but not in the uterine-vessels-ligated group. These results suggest that maternal malnutrition may play an important role in modulation of postnatal drug metabolism pattern of progeny.

Influence of perinatal nutrition on hepatic drug metabolism in the adult rat.

Pregnant rats were fed high-fat (HF, 35% calories) or low-fat (LF, 5% calories) diet from 18 days of gestation to the end of the suckling period. Thereafter, male progeny were fed stock diet for 6 months, then sacrificed or challenged for 3 days with HF diet. There were no immediate posttreatment effects at 30 days of age on liver microsomal cytochrome P-450 or b5, aminopyrine (AP) N-demethylase or benzo(a)pyrene (BP) hydroxylase. At 7 months, cytochrome P-450 was lower in LF-fed than in HF-fed animals. High-fat challenge reduced cytochrome P-450 and cytochrome b5 content in both groups, AP N-demethylase only in LF animals and BP hydroxylase only in HF animals. These differential effects of perinatal exposure to diet on the later response of microsomal mixed-function oxidases to fat challenge suggest that early dietary experience may regulate the pattern of drug metabolism in adult life.

Delayed effects of drug exposure during pregnancy: reproductive function.

This article reviews the delayed effects of anticonvulsants (phenobarbital, phenytoin, and valproate) administered in utero upon the reproductive function in the offspring. Experimental data from the authors' laboratories are discussed in light of this context. Furthermore, comments are made emphasizing the gaps in our knowledge concerning the problem of drug exposure during pregnancy and the complex nature of drug effects upon the reproductive system.

Influence of dietary carbohydrates (alpha-saccharides) on hepatic drug metabolism in male rats.

Young male rats (SD, CD strain) were fed semisynthetic isocaloric diets ad lib for different time periods (3,7,14, or 28 days); both carbohydrate (starch or sucrose) content and fat content were varied. High starch (HST) diet contained starch (73% of calories), corn oil (6%), and casein (21%); low starch (LST) diet contained 6, 73, and 21% of calories, respectively. In high sucrose (HS) or low sucrose (LS) diets, starch was replaced by sucrose. Rats fed LST and LS diets had decreased liver weight compared to those fed HST and HS diets, while liver microsomal protein content (mg/gm liver) was the same in all groups. Significant decreases in microsomal cytochrome P-450 from the basal level were observed in all diets over the period of experimental feeding. This decrease was more prominent with HST or HS diets compared to LST or LS dietary groups. HS diet feeding produced this decrease in cytochrome P-450 levels by 3 days; however, animals on HST diet required 7 days of feeding before they experienced a similar decrease in cytochrome P-450 levels. At 14 days, HST-fed animals had 52% lower liver microsomal cytochrome P-450 than did LST-fed animals. HS-fed animals had 36% lower cytochrome P-450 than LS-fed at 28 days. Similar results were observed for dietary effects on cytochrome b5. Aminopyrine demethylase activity decreased steadily on all diets. p-Nitrophenol glucuronidation was significantly increased in all dietary groups after 2 weeks of diet feeding. These results suggest that dietary carbohydrates and fat (particularly the relative quantities of carbohydrate and fat) may significantly influence the hepatic drug-metabolizing enzymes. It is speculated that these changes may occur due to alteration in the phospholipid composition of endoplasmic reticulum or by limiting the supply of cofactor(s) necessary for optimal mixed function oxidation and conjugation.

Endotoxin and low protein diet induced depression of the rat hepatic drug metabolism.

The effects of the combination of low protein diet feeding and endotoxin (E. coli, serotype 026 B6) upon rat hepatic microsomal mixed function oxidase (MFO) enzymes were investigated. Short-term (7 days) feeding of low protein (8%) diet and acute (single dose) exposure to endotoxin resulted in an additive decrease in MFO enzymes. However, chronic (7 days) endotoxin exposure did not depress liver microsomal MFO enzyme activities except for aniline hydroxylase. Long-term (105 days) feeding of the low protein diet and acute endotoxin exposure further decreased aminopyrine N-demethylase and benzo(a)pyrene hydroxylase activities compared to individual treatments. These results suggest that, under these experimental conditions, the two host-related environmental factors interact and potentiate a decrease in rat hepatic microsomal drug metabolizing enzymes. These observations may be of clinical relevance to explain altered drug reactions in patients with gram-negative infections and endotoxemia under the conditions of malnutrition.

Inhibition of hepatic monooxygenases in neonatal rats by thyroxine.

The effect of thyroxine administration on the activities of hepatic microsomal monooxygenases were studied in neonatal rats. While there were no sexual differences in the activities of hepatic hexobarbital hydroxylase, cytochrome P-450 and cytochrome b5 in 8 day old rats, thyroxine administration significantly reduced the activities of these monooxygenases in both male and female neonates. The results suggest that the liver of the neonatal rat has not developed the ability to respond to the enzyme-stimulating effects of thyroxine as reported for adults.

Effects of endotoxin upon rat hepatic microsomal drug metabolism in vivo and in vitro.

1. Bacterial endotoxin, a soluble lipopolysaccharide, has been studied to ascertain its effects in vivo and in vitro on the hepatic drug-metabolizing enzymes of adult male and female rats. 2. 24 h after a single 1 X 0 or 2 X 0 mg/kg i.p. dose of endotoxin, hexobarbital sleeping time was significantly increased in adult male rats. Significant inhibition of liver microsomal cytochrome P-450 occurred after 6 h and continued only until 24 h after endotoxin administration, while injection of inactivated endotoxin did not result in any significant decrease of hepatic mixed-function oxidase enzymes or cytochrome P-450. In contrast, rho-nitrophenol-UDP-glucuronyltransferase enzyme activity was unaffected by these levels of endotoxin. 3. Electron-microscopic examination of rat liver hepatocytes did not reveal any significant change in ultrastructure 24 h after a single i.p. dose of endotoxin. 4. Endotoxin failed to depress the phenobarbitone- or 3-methylcholanthrene-induced forms of cytochrome P-450 and the dependent mono-oxygenase enzymes. Simultaneous administration of phenobarbital and endotoxin resulted in 100% mortality of rats. Combination of 3-methylcholanthrene and endotoxin did not block the induction of cytochrome P-448 or dependent benzo[a]pyrene hydroxylase activity. 5. Addition of endotoxin in vitro resulted in significant inhibition of hepatic microsomal cytochrome P-450 and aminopyrine N-demethylase activity only on preincubation with an NADPH-generating system supplemented with EDTA.

Perinatal development of N-acetyltransferase in hepatic and extrahepatic tissues of guinea pigs.

Hepatic and extrahepatic distribution and the perinatal developmental pattern of p-aminobenzoic acid (PABA)-N-acetyltransferase (E.C.2.3.1.5) in guinea pig liver, lung, and placenta were studied. In adult guinea pigs, kidney and small intestine enzyme specific activities were equivalent to hepatic activity. Lung enzyme activity was about 15% of that in adult liver. No sex differences in hepatic or extrahepatic distribution of enzyme levels were evident. The perinatal development study revealed that fetal as well as neonatal animals are capable of N-acetylation. The peak in liver and lung activity occur between 3 and 8 days after birth. Placenta has about 50% of adult liver activity for PABA-N-acetylation and it declines near term. These data indicate that extrahepatic organs of guinea pigs significantly contribute to PABA-N-acetylation.

Susceptibility of mice to phenytoin-induced cleft palate correlated with inhibition of fetal palatal RNA and protein synthesis (41255).

Phenytoin administered to pregnant mice during the critical embryonic period of palatal differentiation produced 50% cleft palates in the Ajax (A/J) strain compared to 1.6% clefts in the C57BL/6 (B6) strain of mice. Furthermore, a single maternal injection of phenytoin produced a significantly greater and more persistent decrease in fetal palatal RNA and protein synthesis in the sensitive A/J strain compared to that in the insensitive B6 strain of mice. Thus, these differential effects of phenytoin on RNA and protein synthesis are associated with the differential susceptibility to the teratogenic action of phenytoin in the two strains.

Postnatal development of mixed-function oxidation and conjugation in hemoglobin-free perfused rat liver.

In perfused livers from normal developing rats, rates of p-nitroanisole O-demethylation, nearly undetectable at 8 days of age, increased progressively to a maximum of 5.1 mumol of p-nitrophenol formed per gram of liver wet weight per hour by 20 days of age. However, by 30 days of age, the rate of p-nitrophenol formation decreased to 2.8 mumol/g/h, in spite of a 300% increase in microsomal p-nitroanisole O-demethylase activity between 20 and 30 days of age. Conjugation of p-nitrophenol in perfused livers was minimal before 16 days of age. Subsequently, rates of conjugation of p-nitrophenol increase in parallel with the development of hepatic glycogen metabolism and uridine diphosphate-glucuronyltransferase activity. At all stages of development studied (8-30 days), phenobarbital treatment increased the liver weight, cytochrome P-450 content and and in vitro p-nitroanisole O-demethylase and glucuronyltransferase activities. In perfused livers from phenobarbital-treated pups, mixed-function oxidation and conjugation increased in parallel with the development of the microsomal enzymes. These data indicate that both the development of microsomal enzyme activity and the availability of required cofactors (e.g. NADPH) influence rates of mixed-function oxidation and conjugation in intact liver during development.

Altered thyroid hormone status and dichlorvos toxicity in rats.

The effects of altered thyroid function upon serum cholin-esterase levels and dichlorvos toxicity were studied in adult male and female rats. No sexual dimorphism was observed in the base level of serum cholinesterase, however, oral administration of dichlorvos (52.5 mg/kg) resulted in 30% mortality to intact female rats. Hypothyroidism significantly elevated while hyperthyroidism decreased serum cholinesterase enzyme activity in both sexes of rats. Hyperthyroid females were more susceptible than males to dichlorvos challenge even with similar decrease in serum cholinesterase activity.

Gram-negative endotoxin administration decreases hepatic drug-metabolizing enzymes during development in rats.

The frequency of gram-negative infections and endotoxemia in the perinatal period prompted an investigation of the effects of endotoxin (Escherichia coli 026B6) on hepatic drug metabolism. Gravid female rats given injections IP with different dosages of lipopolysaccharide during late pregnancy resulted in significant depression of the liver microsomal cytochrome P-450 dependent monooxygenase activities. The acute administration of endotoxin to mothers (1.4 mg/kg on seventh day after parturition) significantly decreased the hepatic activity of aminopyrine demethylase and contents of cytochrome P-450 of suckling neonates and mothers. However, chronic administration of endotoxin (0.2 mg/kg/day for 7 days) to lactating mothers did not alter neonatal enzyme activities. When neonates themselves were given injections of endotoxin (1.0 mg/kg) at 7, 16, and 27 days of age, a significant reduction in levels of mixed function oxidase enzymes was observed. These observations suggest that the ability of mothers and neonates to metabolize drugs is significantly decreased upon exposure to endotoxin, and this demands careful evaluation of drug disposition studies in gram-negative sepsis.

Phenobarbital exposure in utero: alterations in female reproductive function in rats.

Phenobarbital administration to pregnant rats from day 12 to day 19 of gestation suppressed body weight gain and produced significant effects on reproductive function in their offspring. These effects included delays in the onset of puberty, disorders in the estrous cycle, and infertility. Moreover, the animals exposed to phenobarbital in utero showed altered concentrations of sex steroids, gonadotrophic hormones, and estrogen receptors. These findings suggest that phenobarbital treatment during prenatal development can produce permanent alterations in sexual maturation.