Dibromoacetic acid, a prevalent by-product of drinking water disinfection, compromises the synthesis of specific seminiferous tubule proteins following both in vivo and in vitro exposures.

Dibromoacetic acid (DBA) is a by-product of drinking water disinfection that alters spermatogenesis in adult male rats. To identify a mechanism by which DBA alters spermatogenesis, seminiferous tubules representing specific groups of spermatogenic stages were exposed either in vivo or in vitro, and structural and functional consequences were evaluated. Seminiferous tubules representing stages I-V, VI-VIII, and IX-XIV were isolated from testes of adult rats and cultured overnight in conditions of reduced oxygen and temperature. For in vivo exposures, seminiferous tubules were recovered from animals that had received 250 mg/kg DBA via gavage for 5 days. For in vitro exposures, 180 and 600 microM concentrations were tested; these concentrations bracket the concentration of DBA observed within the testis following in vivo exposure. Protein synthesis was evaluated by 35S-methionine labeling overnight and quantitative analysis of radiolabeled proteins in mini, 2-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Radio-inert cultures were processed for light and electron microscopy. Morphologicaf evaluation indicated that all spermatogenic stages of the seminiferous tubules from control animals were well maintained during the isolation and culture period. Although no treatment-related lesions were observed following in vivo exposure, histological alterations were observed at the lowest in vitro exposure. There was a significant diminution (P < .05) in the synthesis of 4 cytosolic proteins following both in vivo and in vitro exposures. Diminution in these proteins was restricted to stages I-V and IX-XIV of spermatogenesis, suggesting that proteins involved in the early stages of spermiogenesis are uniquely sensitive to DBA exposure. Because histology and protein synthesis were affected by relevant in vitro exposures, this indicates that DBA is capable of altering spermatogenesis directly.

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).

Influence of the drinking water disinfection by-product dibromoacetic acid on rat estrous cyclicity and ovarian follicular steroid release in vitro.

The drinking water disinfection by-product, dibromoacetic acid (DBA) has been reported to affect gonadal functions in the male rat. However, there is little information regarding the influence of DBA on female reproductive activity. Consequently, the present study investigated the effects of DBA on estrous cyclicity and the impact in vitro of DBA on ovarian follicular steroid secretion. Regularly cycling animals were dosed with DBA (0 to 270 mg/kg/day) for 14 days and estrous cyclicity was monitored during treatment and for an additional 2-week posttreatment interval. A dose-related alteration in cyclicity was observed at 90 and 270 mg/kg/day, which persisted through the posttreatment monitoring in the high dose group. An in vitro exposure of preovulatory follicles to DBA was then used to assess the influence of DBA on steroid release. To select a concentration for use, a single oral exposure to 270 mg/kg was administered, and the mean blood levels were determined over a 5-h interval. For this in vitro work, pairs of preovulatory follicles from PMSG-primed immature rats were exposed to 0 or 50 microg/mL DBA over a 24-h period and evaluated for estradiol and progesterone release under baseline and hCG-stimulated conditions. The influence of tumor necrosis factor (TNFalpha) exposures under these conditions was also determined. In the nonstimulated condition, DBA was found to increase the release of estradiol, but had no detectable effect in response to hCG. Progesterone, however, showed marked suppression under hCG stimulation following exposure to DBA, while nonstimulated secretion was unaffected. TNFalpha by itself also suppressed stimulated progesterone release, but had no additional effect in combination with DBA. The data suggest that one factor in the disruption in estrous cyclicity could be an alteration in steroid production, which was characterized by separate effects on both estradiol and progesterone secretion.

Sample preparation and high-performance liquid chromatographic analysis of deoxyribonucleoside triphosphates in individual rat embryos.

A rapid, robust, and sensitive method has been developed to measure concentrations of deoxyribonucleoside triphosphates in individual, day 14 rat embryos by modifying and optimizing existing methods for cellular extracts. Significant changes include: (i) oxidative degradation of ribonucleoside triphosphates using methylamine at lower pH (decreased from 6.5 to 4.0) to improve poor HPLC peak shape of early eluting nucleotides; (ii) glass fiber disc solid-phase extraction of the reaction mixture, which dramatically reduces impurities that interfere with nucleotide measurement, eliminates the necessity of column regeneration, and allows mobile phase recycling; and (iii) lower ionic strength (reduced from 0.4 to 0.26 or 0.12 M ammonium phosphate) and higher pH (increased from 3.25 to 5.55 or 6.98, respectively) mobile phase, conditions which are less destructive to the column's bonded phase and silica support, thereby contributing to longer column life. Enhancements include: (i) filtration of the sample prior to HPLC injection and addition of an in-line filter, guard column, and saturating precolumn of silica in the mobile phase flow, which aids substantially in extending column life and improves chromatographic stability, and (ii) inclusion of an internal standard to correct for mechanical losses. Limits of determination at a signal to noise ratio of 6:1 range from 5.5 to 12 pmol on-column or 0.41 to 0.87 pmol/mg of embryonic tissue depending on the specific nucleotide. Recoveries are quantitative for all nucleotides, and interassay variabilities are between 5 and 7% when quantified by peak height. The method has also been applied successfully to analysis of murine erythroleukemic cell cultures and this, when coupled with the embryo results, suggests its general utility.

Nucleoside-mediated mitigation of 5-fluorouracil-induced toxicity in synchronized murine erythroleukemic cells.

5-Fluorouracil (5-FU) is a chemotherapeutic agent known to retard embryonic growth and induce cleft palate and limb deformities. The predominant mechanism underlying its toxic action is thought to be inhibition of thymidylate synthetase (TS), and hence thymidine triphosphate (dTTP) synthesis, resulting in alteration of the balance of deoxynucleotide (dNTP) pools and disruption of DNA synthesis. Indeed, previously we demonstrated retarded cell-cycle progression concurrent with a 60% decrease in TS activity in rat whole embryos following maternal exposure to 40 mg/kg 5-FU on Gestational Day 14 and in the murine erythroleukemic cell (MELC) suspension culture following exposure to 5-25 microM 5-FU for 2 hr. In the study described herein, we used high-performance liquid chromatography (HPLC) to demonstrate in both of these model systems that 5-FU exposure results in similar patterns of dNTP perturbations: a prolonged decrease in dTTP and dGTP levels and an increase in dCTP and dATP. In addition, we used centrifugal elutriation to synchronize MELC in the phases of the cell cycle (G0/G1 and early S) most sensitive to 5-FU to investigate the ability of nucleoside supplementation to mitigate 5-FU-induced toxicity. Our data indicate that following a 2-hr exposure to 5-25 microM 5-FU, supplementation with 1-10 microM thymidine (TdR) for 24 hr partially reverses 5-FU-induced toxicity as evidenced by increased cellular proliferation and cell-cycle progression and amelioration of 5-FU-induced perturbations of protein synthesis and cellular membrane permeability compared to unsupplemented 5-FU-exposed cells. However, TdR concentrations >/=100 microM inhibited growth or were cytotoxic. In comparison, supplementation with 10 microM-10 mM of deoxycytidine (CdR) was not toxic, but effected a dose-dependent recovery from 5-FU-induced toxicity. At 1-100 microM, neither deoxyadenosine nor deoxyguanosine supplementation reduced 5-FU-induced toxicity; at higher concentrations, both purine nucleotides inhibited cell growth. Although these results support the hypothesis that 5-FU disrupts the MELC cell cycle by depleting dTTP (a perturbation that is reversible by TdR supplementation), they also indicate that CdR supplementation offers an additional recovery pathway.

Critical periods of sensitivity to the developmental toxicity of inhaled methanol in the CD-1 mouse.

Exposure of pregnant CD-1 mice to methanol (MeOH) by inhalation on gestation days (gd) 6-15 results in dose-related increases in fetal cleft palate, exencephaly, and skeletal defects. Here, critical periods for the developmental toxicity of MeOH were assessed in pregnant CD-1 mice exposed to 10,000 ppm MeOH or filtered air for 7 hr/day on 2 consecutive days during gd 6-13, or to single day (7 hr) exposures to 10,000 ppm MeOH during gd 5-9. Mice received water but not food during exposure. Maternal blood MeOH was determined at times during, at the end of, and subsequent to a single 7 hr exposure on gd 7. On gd 17, remaining mice were weighed, killed, and gravid uteri removed. Live, dead, and resorbed fetuses were counted, and live fetuses were examined, weighed, and preserved in 70% ethanol. All fetuses were examined externally and for cleft palate, eviscerated, and stained with Alizarin red for skeletal examination. Pregnant mice lost an average of 0.3-2.9 g during 7 hr exposure to either filtered air or MeOH, but a MeOH treatment effect was evident only with 2-day exposure on gd 7-8. Peak maternal blood MeOH concentration (at the end of exposure) was approximately 4 mg/ml, and MeOH was cleared from maternal blood within 24 hr. Some fully resorbed litters were observed with 2-day MeOH exposures on gd 6-7 or 7-8, or 1-day exposure on gd 7. With 1-day MeOH exposure on gd 7, the number live was lower than with exposure on any other day. As previously reported, cleft palate, exencephaly, and skeletal defects were the fetal anomalies observed in this mouse strain. Cleft palate occurred with 2-day exposures on gd 6-7 through gd 11-12 (peak on gd 7-8), and with 1-day exposure on gd 5 through gd 9 (peak on gd 7). Exencephaly occurred with 2-day exposures on gd 6-7 through gd 8-9 (peak gd 6-7) or 1-day exposure on gd 5 through gd 8 (peak on gd 7). Skeletal elements malformed included the exoccipital (peak gd 6-7, gd 5), atlas (peak gd 6-7, gd 5,6), axis (peak gd 6-7, gd 7), cervical vertebra 7 with a rib (peak gd 6-7, gd 7), and lumbar vertebra 1 with a rib (peak gd 7-8, gd 7). An increased incidence of fetuses with 25 presacral vertebrae (normal = 26) was observed with methanol exposure on gd 5, whereas an increased incidence of fetuses with 27 presacral vertebrae was observed with MeOH exposure on gd 7. These results indicate that gastrulation and early organogenesis represent a period of increased embryonal sensitivity to methanol.

Assessment of offspring development and behavior following gestational exposure to inhaled methanol in the rat.

The prospect of widespread human exposure associated with its use as an alternative fuel has sparked concern about the toxic potential of inhaled methanol (MeOH). Previous studies have revealed congenital malformations in rats following inhaled MeOH (Nelson et al. (1985). Fundam. Appl. Toxicol. 5, 727-736) but these studies did not include postnatal behavioral assessment. In the present study, pregnant Long-Evans rats were placed in exposure chambers containing 15,000 ppm MeOH or air for 7 hr/day on Gestational Days (GD) 7-19. The total alveolar dose of methanol was estimated at about 6.1 g/kg/day, for a total dose of about 42.7 g/kg for the entire study. Maternal body weights were recorded daily and blood methanol concentrations were determined at the end of exposure on GD 7, 10, 14, and 18. Following birth (Postnatal Day 0 [PND 0]), a number of tests were performed at various points in development, including: offspring mortality and body wt (PND 1,3), motor activity (PND 13-21, 30, 60), olfactory learning (PND 18), behavioral thermoregulation (PND 20-21), T-maze learning (PND 23-24), acoustic startle response (PND 24, 60), reflex modification audiometry (PND 60), pubertal landmarks (PND 31-56), passive avoidance (PND 72), and visual-evoked potentials (PND 160). Maternal blood MeOH levels, measured from samples taken within 15 min after removal from the exposure chamber, declined from about 3.8 mg/ml on the first day of exposure to 3.1 mg/ml on the 12th day of exposure. MeOH transiently reduced maternal body wt (4-7%) on GD 8-10, and offspring BW (5%) on PND 1. No other test revealed significant effects of MeOH. Prenatal exposure to high levels of inhaled MeOH appears to have little effect on this broad battery of tests beyond PND 1 in the rat.

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)

Effect of inhaled methanol on pituitary and testicular hormones in chamber acclimated and non-acclimated rats.

Two experiments were conducted in which the acute effects of inhaled methanol on serum hormones associated with reproductive function in the male rat were evaluated. In the first experiment, rats exposed to methanol (0, 200, 5000 and 10,000 ppm) for 6 h were killed at the end of the exposure period (6 h) or the following morning (24 h). Also, because the process of exposure itself could modify neuroendocrine function, the effect of the handling associated with placing the rat in the exposure chamber was evaluated further by dividing the exposed animals into acclimated (2 weeks of prior handling) and non-acclimated groups. At 6 h, an effect of prior handling was noted in the sham-exposed rats, with serum luteinizing hormone (LH) of the non-acclimated group being greater than that of the acclimated group. Serum LH concentrations were altered by methanol exposure, but the direction of change and the exposure level at which an effect was noted differed between the acclimated and non-acclimated rats. Methanol (5000 ppm) reduced serum LH in the non-acclimated animals, while 10,000 ppm increased LH in the acclimated rats. Follicle stimulating hormone (FSH) and testosterone were unchanged by methanol in rats killed at 6 h. Thus, this experiment did not confirm earlier reports that exposure to 200 ppm for 6 h reduced serum testosterone. At 24 h, an effect of prior handling was still present in the hormonal measures, with serum and interstitial fluid testosterone concentrations being greater in the non-acclimated rats. Also, there was a dose x handling interaction with methanol exposure inducing an increase in serum testosterone in the non-acclimated rats (up to 5000 ppm) and a decrease in the acclimated rats (up to 10,000 ppm). In the second experiment, groups of acclimated and non-acclimated rats were exposed to 0 or 5000 ppm methanol for 1, 2 and 6 h and killed immediately after removal from the chamber. Serum LH, testosterone and FSH values were not different in sham- vs methanol-exposed rats at any time point. As in experiment 1, an effect of prior handling was noted. In general, the concentrations of these hormones and serum prolactin in the non-acclimated rats were greater than those observed for acclimated rats. Methanol exposure resulted in increased prolactin concentrations under both handling conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

High-performance liquid chromatographic assay of biphenyl metabolism by hepatocytes cultured in an embryo/hepatocyte co-culture medium.

A high-performance liquid chromatographic method has been modified for the evaluation of both Phase I and II metabolism of biphenyl by hepatocytes maintained in an embryo/hepatocyte co-culture medium. Extracts of the media, before and after hydrolysis of conjugates, are directly injected onto the HPLC and the major hydroxylated metabolites plus unmetabolized biphenyl are detected by fluorescence after separation under gradient or isocratic conditions. The method is almost free of interferences and is relatively simple and rapid. In the case of the monohydroxylated derivatives, the minimum media concentrations which can be measured are 7 to 20 nM (0.07 to 0.2 pmol on-column). Recoveries from culture medium to which known amounts of biphenyl and metabolites had been added were quantitative (90-103%) and the reproducibility good (interassay CV less than 5%). The assay was applied to cultures of hepatocytes derived from rabbit and from phenobarbital induced and noninduced rat.

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.

Effect of ozone on serum lipids and lipoproteins in the rat.

Exposure of male rats to 0 (air), 1, 1.75, and 3 ppm ozone (O3) 5 hr/day for a total of 10 days resulted in a positive linear relationship between ozone concentration and the concentrations of serum total lipoprotein free cholesterol (FCh) and high-density lipoprotein total cholesterol (HDL-Ch). The latter response was reflected in both its free (HDL-FCh) and esterified (HDL-ChE) components. On the other hand, serum triglycerides (TG) showed a marked decreasing linear trend with increasing ozone concentration. As judged by decreased body weights with no accompanying differences in feed consumption, apparent metabolic rate increased as ozone concentration increased. In another experiment, male rats were exposed 5 hr/day to either air or 1 ppm O3 for a total of 15 days. Groups of animals from each exposure were sampled at times ranging from immediately after to 44 hr postexposure. In agreement with the concentration response study, effects of O3 included increases in serum total cholesterol (Ch), HDL-Ch and HDL-FCh, and a decrease in TG. In addition, the degree of effects appeared to be maintained over the 44-hr period and to be greater than that observed at 1 ppm O3 in the concentration-response study.

Influence of ozone on pentobarbital pharmacokinetics in mice.

Previous studies have indicated that acute exposure to ambient concentrations of ozone (O3) as low as 196 micrograms/m3 (0.1 ppm) increases pentobarbital (PEN)-induced sleeping time in female mice. To elucidate potential mechanisms involved, additional studies were performed. A 3 h exposure to 9800 micrograms O3/m3 (5 ppm) did not affect brain concentrations of PEN at time of awakening, even though sleeping time was increased. Exposure for 3 h to 9800 micrograms O3/m3 (5 ppm) did not alter the pattern of brain or plasma metabolites of PEN. Pentobarbital clearance followed first-order kinetics with a one-compartment model. Mice exposed to 9800 micrograms O3/m3 (5 ppm) for 3 h had a 106% increase in the plasma half-life of pentobarbital; at 1960 micrograms O3/m3 (1 ppm) for 3 h, a 71% increase was observed. It therefore appears possible that PEN-induced sleeping time might be increased due to an decrease in hepatic metabolism of PEN.

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.

Constituents of Cannabis sativa L. VIII: Possible biological application of a new method to separate cannabidiol and cannabichromene.

Synthetic and naturally occurring cannabidiol and cannabichromene were distinctly separated without derivation by GLC using a 6% OV-1 column; an artifact of cannabichromene, cannabicyclol, was separated from (minus)-delta9-trans-tetrahydrocanna-bivarin. This procedure is versatile and applicable for the quantitation of Cannabis containing both cannabidiol and cannabichromene. Biological interaction among (minus)-delta9-trans-tetrhydrocanabinol, cannabichromene, and other cannabinoids in natural Cannabis preparations can now be studied. In the phenyl methyl silicone polymer series, cannabidiol precedes ca-nabichromene on columns containing below a 50% phenyl-to-methyl ratio. Columns containing below a 50% phenyl-to-methyl ratio. Columns containing a 50:50 or greater ratio of phenyl to methyl reverse the separation order with cannabichromene preceding cannabidiol.