Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. II: Effects on conjugating enzymes.

The effects of long-term caloric restriction on the hepatic phase II drug metabolizing enzymes were investigated in the male Fischer 344 rat. Rats that had been restricted to 60% of their pair-fed control consumption from 14 weeks post-partum exhibited altered conjugating enzyme activities at 22 months. Caloric restriction significantly reduced the age-related decrease in glutathione-S-transferase activity towards 1,2-dichloro-4-nitrobenzene, but did not significantly alter the age-related changes in UDP-glucuronyltransferase or sulfotransferase activities towards hydroxysteroids. Caloric restriction appeared to increase hepatic microsomal UDP-glucuronyltransferase activity toward bilirubin and gamma-glutamyltranspeptidase activities. These observations suggest that caloric restriction has multiple effects on the hepatic phase II drug metabolizing enzymes in the rat. Such effects may alter hepatic metabolism and activation or detoxification of drugs and carcinogens.

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. I: The cytochrome P-450 dependent monooxygenase system.

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6 beta-hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad libitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn1&2, P-452 and P450j may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.

Ames assays and unscheduled DNA synthesis assays on 2, 4-dichlorophenoxyacetic acid and its derivatives.

2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes. In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D. There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays. These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats.

In vivo micronucleus assays on 2,4-dichlorophenoxyacetic acid and its derivatives.

The potential for 2,4-D and seven of its salts and esters to induce cytogenetic abnormalities in mammalian cells in vivo was investigated in the mouse bone marrow micronucleus test. All the test materials were administered to male and female mice by oral gavage and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow were determined at intervals of 24, 48 and 72 h following dosing. There were no significant increases in the incidence of MN-PCE in the treated mice at any of the bone marrow sampling times. These results are consistent with the reported lack of in vitro genetic toxicity for these materials in various in vitro genotoxicity assays as well as the absence of carcinogenic potential for 2,4-D in both mice and rats.

Comparison of a modification of the ellman method to measure carbamate inhibition of acetylcholinesterase in plasma, erythrocytes and brain tissues in sprague dawley rats using two analytical systems.

Inhibition of the enzyme acetylcholinesterase (AChE) using a carbamate compound was measured in 30 Crl: CD(R)BR Sprague Dawley rats. Erythrocyte, plasma, and brain tissues were analyzed using modifications of the Ellman technique(1) on two different clinical chemistry analyzers. Both EDTA and heparin anticoagulated whole blood were used for the erythrocyte and plasma tests. Results demonstrated similar inhibition of the enzyme in all three tissues between the control and dosed groups using the two technique modifications and instruments. Final inhibition of plasma and erythrocyte AChE for the control vs. treated groups (males and females combined) was 89.5% vs. 82% and 39% vs. 38% for the Technicon AutoAnalyzertrade mark vs. the Boehringer Mannheim Hitachitrade mark 704, respectively. Inhibition of the left and right brain segments for the control vs. treated groups (males and females combined) was 35% vs. 39% and 33.2% vs. 29% for the Technicon and the Hitachi, respectively. All inhibitions were significant at the 5% level using two tailed Dunnett's t-Test. Hemolysates prepared from EDTA whole blood packed cells gave more consistent results on the Hitachi 704.

Stereoselective behavioral effects of the isomers of pentobarbital and secobarbital in the pigeon.

Little information is available on the contribution of the isomers of pentobarbital and secobarbital to the behavioral effects of the racemic mixtures. To determine the contribution of each isomer on food-maintained behavior, pigeons were trained to respond under either a multiple fixed-ratio 30, fixed-interval 600 (mult FR30 FI600) schedule, or a multiple fixed-ratio 30, variable-interval 60-sec (mult FR30 VI60) schedule under which every response under both the FR and VI components also produced a brief electrical shock (punishment). Dose-response curves were determined separately for each isomer of pentobarbital (1-17.5 mg/kg i.m.) and secobarbital (1-17.5 mg/kg i.m.). Under the mult FR30 FI600 schedule, the S-(-)-isomers of pentobarbital and secobarbital were slightly less than 2-fold more potent than the R-(+)-isomers, but both isomers produced qualitatively similar effects on responding. The S-(-)-isomers of both barbiturates produced increases in punished responding under the mult FR30 VI60 at lower doses than the R-(+)-isomers. At high doses (greater than 10 mg/kg), the S-(-)-isomers suppressed responding early in the session resulting in a lower average rate of punished responding for the session than the R-(+)-isomers. The peak increase in punished responding was observed after 10 mg/kg of the R-(+)-isomers of pentobarbital and secobarbital. These results indicate that the effect of the isomers of pentobarbital and secobarbital on mult FR30 FI600 responding and on suppressed responding are qualitatively similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal disposition of delta 9-tetrahydrocannabinol (THC) during late pregnancy in the rhesus monkey.

Three late-term (Gestational Days 146-151) rhesus monkeys were given 0.3 mg/kg delta 9-tetrahydrocannabinol (THC) intravenously via the maternal radial vein to quantify the placental transfer and fetal disposition of THC, the major psychoactive component of marijuana. Simultaneous blood samples were obtained from a maternal uterine vein and an intraplacental artery at 0, 3, 15, 30, 45, 60, 90, 120, and 180 min after dosing using an intraplacental cannulation technique. Samples of fetal plasma, spleen, testis, lung, brain, liver, bile, kidney, adrenals, thymus, and placenta were obtained at 180 min postdose. Samples were analyzed for THC and a major metabolite, 11-nor-9-carboxy-THC (11-nor), by radioimmunoassay (RIA). Peak plasma THC values were obtained 3 and 15 min after dosing in the mother (1419 ng/ml) and fetus (83 ng/ml), respectively. By 3 hr, maternal and fetal plasma THC levels were equal (37 ng/ml). Maternal plasma was sampled beyond 180 min at 24, 48, 72, and 96 hr postdose, times at which THC and 11-nor concentrations were either near or at the lower limit of sensitivity for the RIA (2 ng/ml). While maternal plasma 11-nor levels peaked at 1 hr (44 ng/ml), almost no 11-nor was detected in fetal plasma (less than 5 ng/ml). Fetal tissue concentrations of THC were 53 +/- 6 ng/g (SE) for brain and 114 +/- 10 ng/g for liver, while 11-nor was undetectable in placenta, fetal liver, and fetal brain. These data demonstrate that THC rapidly crosses the placenta and enters the fetus. The lack of 11-nor in fetal plasma and tissues suggests that this metabolite does not readily cross the placenta and that the fetus does not readily metabolize THC to 11-nor at this stage of development. A portion of this data was presented at the 1985 meeting of The American Society of Primatologists and at the 1986 meeting of The Teratology Society.

Comparative subchronic studies on 2,4-dichlorophenoxyacetic acid, amine, and ester in rats.

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Subchronic toxicity studies in rats were conducted on three forms of 2,4-D: the parent form, 2,4-D acid; 2,4-D dimethylamine salt (DMA); and 2,4-D 2-ethylhexyl ester (2-EHE). Doses in the subchronic studies (on an acid equivalent basis) were 0, 1, 15, 100, and 300 mg/kg/day. Major treatment related findings in the three studies included decreases in red cell mass, decreases in T3 and T4 levels, decreases in ovary and testes weights, increases in liver, kidney, and thyroid weights, and cataracts and retinal degeneration (high-dose females). These data demonstrated the comparable toxicities of 2,4-D acid, DMA, and 2-EHE and support a subchronic no-observed-effect level of 15 mg/kg/day for all three forms. In summary, the findings of these studies indicate comparable low subchronic toxicity potentials among representative forms of 2,4-D.

Single-dose and chronic dietary neurotoxicity screening studies on 2,4-dichlorophenoxyacetic acid in rats.

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Single-dose acute and 1-year chronic neurotoxicity screening studies in male and female Fischer 344 rats (10/sex/dose) were conducted on 2,4-D according to the U.S. EPA 1991 guidelines. The studies emphasized a Functional Observational Battery (which included grip performance and hindlimb splay tests), automated motor activity testing, and comprehensive neurohistopathology of perfused tissues. Dosages were up to 250 mg/kg by gavage for the single-dose study, and up to 150 mg/kg/day in the diet for 52 weeks in the repeated-dose study. In the acute study, gavage with 250 mg/kg test material caused slight transient gait and coordination changes and clearly decreased motor activity at the time of maximal effect on the day of treatment (day 1). Mild locomotor effects occurred in one mid-dose rat (75 mg/kg), on Day 1 only. No gait, coordination, or motor activity effects were noted by day 8. In the chronic study, the only finding of neurotoxicologic significance was retinal degeneration in females in the high-dose group (150 mg/kg/day). Body weights of both sexes were slightly less than controls in the mid-dose group, and 10% less than controls in the high-dose group. In summary, the findings of these studies indicated a mild, transient locomotor effect from high-level acute exposure, and retinal degeneration in female rats from high-level chronic exposure. Based on the results from these two studies, the no-observed-adverse-effect level for acute neurotoxicity was 15 mg/kg/day and for chronic neurotoxicity was 75 mg/kg/day.

Subchronic toxicity (90-day) study with para-nonylphenol in rats.

As a component to the risk assessment process for para-nonylphenol (NP; CASRN 84852-15-3), a 90-day study was conducted in rats following U.S. EPA TSCA guidelines and Good Laboratory Practice regulations. NP was administered to four groups of rats at dietary concentrations of 0, 200, 650, or 2000 ppm which corresponded to approximate dietary intakes of 0, 15, 50, or 150 mg/kg/day, respectively. There were 25 rats/sex/group in the control and high-dose groups and 15 rats/sex/group in the low- and middose groups. Ten of the 25 rats/sex in the control and high-dose groups were designated as recovery animals and were maintained on control diets for 4 weeks after completion of the 90-day exposure period to assess the reversibility of any effects which might be observed. To evaluate for the possible weak estrogen-like activity that has been reported for NP in a number of screening assays, estrous cyclicity was monitored using vaginal cytology during Week 8 of the study, and sperm count, motility, and morphology were evaluated at termination. In-life effects from NP exposure were limited to small decreases in body weight and food consumption in the 2000-ppm dose group. Postmortem measurements at Week 14 indicated a dose-related kidney weight increase in males and a decrease in renal hyaline globules/droplets in males from the high-dose group. The kidney weights showed complete recovery following the 4-week postdosing recovery period. Due to the small magnitude of the changes (i.e., all weights were within or near laboratory historical control values) and the lack of correlating clinical or histopathological changes, the kidney weight alterations were not considered toxicologically significant. The biological significance of reduced hyaline in the kidneys of male rats from the high-dose group is uncertain. Renal tubular hyaline is associated with the rat-specific protein, alpha-2u-globulin, and, therefore, this finding was not considered toxicologically relevant to humans. No other effects attributable to NP were observed. No changes were observed for estrous cycling, sperm evaluations, or effects on endocrine organs. NP, therefore, did not manifest any estrogen-like activity as measured in these parameters at dietary concentrations as high as 2000 ppm, the maximum dose administered in this study. Based on the minor findings for the 2000-ppm dose group, the NOAEL (no-observed-adverse-effect level) for NP in this study is considered to be 650 ppm in the diet, corresponding to an approximate intake of 50 mg/kg/day.

Comparative subchronic and chronic dietary toxicity studies on 2,4-dichlorophenoxyacetic acid, amine, and ester in the dog.

Forms of 2,4-dichlorophenoxyacetic acid (2,4-D) are herbicides used in the control of a wide variety of broadleaf and woody plants. Subchronic toxicity studies in dogs were conducted on three forms of 2,4-D: the parent form, 2,4-D acid (ACID); 2,4-D dimethylamine salt (DMA); and 2,4-D 2-ethylhexyl ester (2-EHE). The three studies were designed to allow for comparison of the toxicity of the three forms. Doses in the subchronic studies, on an acid equivalent basis, were 0, 0.5 (ACID only), 1.0, 3.75, and 7.5 mg/kg/day. Treatment related findings in the three studies included reductions in body weight gain, and food consumption, and minor increases in blood urea nitrogen, creatinine, and alanine aminotransferase. The data from the three subchronic studies demonstrated the comparable toxicity of ACID, DMA, and 2-EHE and support a subchronic no observed adverse effect level (NOAEL) of 1.0 mg/kg/day for all three forms. Due to the similarity in toxicity of the three forms of 2,4-D, a 1-year chronic toxicity study was performed on the parent ACID to fully characterize the potential toxicity of 2,4-D in the dog. ACID was well tolerated at doses of 0, 1.0, 5.0, and 7.5 mg/kg/day. The clinical pathology alterations were similar to those seen in the subchronic studies and were not progressive. The histopathology alterations observed were not severe in nature and the no observed effect level in the chronic study was determined to be 1.0 mg/kg/day. There was no indication of any immunotoxic or oncogenic response in the studies. In conclusion, the findings of these studies indicate comparable toxicity among representative forms of 2,4-D and their generally low toxicity following subchronic and chronic dietary exposure in the dog.

Chronic dietary toxicity/oncogenicity studies on 2,4-dichlorophenoxyacetic acid in rodents.

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Doses in the 2-year chronic/oncogenicity rat study were 0, 5, 75, and 150 mg/kg/day. The chronic toxicity paralleled subchronic findings, and a NOEL of 5 mg/kg/day was established. A slight increase in astrocytomas observed (in males only) at 45 mg/kg/day in a previously conducted chronic rat study was not confirmed in the present study at the high dose of 150 mg/kg/ day. Doses in the 2-year mouse oncogenicity studies were 0, 5, 150, and 300 mg/kg/day for females and 0, 5, 62.5, and 125 mg/ kg/day for males. No oncogenic effect was noted in the study. In summary, the findings of these studies indicate low chronic toxicity of 2,4-D and the lack of oncogenic response to 2,4-D following chronic dietary exposure of 2,4-D in the rat and mouse.