Transplacental and lactational transfer of p,p'-DDE in Sprague-Dawley rats.

p,p'-DDE (hereafter DDE), a persistent metabolite of p,p'-DDT, is a widespread environmental contaminant that can induce antiandrogenic developmental effects in rats. Quantitative measurements of the transfer of DDE from pregnant or lactating dams to the fetus or suckling neonate were performed, and physiologically based pharmacokinetic (PBPK) models for the transplacental and lactational transfer of DDE were developed. Pregnant Sprague-Dawley rats were dosed by gavage in corn oil with either 10 or 100 mg DDE per kg body wt per day from Gestation Day (gd) 14 to 18. DDE was analyzed in several maternal tissues as well as in fetal and neonatal tissues from gd 15 to Postnatal Day (pnd) 21. Fetal DDE concentrations were about threefold lower than corresponding placental concentrations. By adopting a cross-fostering design, the contributions of transplacental and lactational transfer were compared. In the pup liver, where DDE was detectable in the 100 mg/kg groups on pnd 10, the lactationally exposed group had DDE concentrations about 50 times higher than those of the in utero only exposure group; the lactation only exposure groups had DDE tissue dose profiles very similar to those of the in utero plus lactation exposure groups, indicating that the lactational route is far more important than the in utero route quantitatively. The PBPK models postulated initial absorption of DDE into both the blood circulation and lymphatic system with the primary storage sites being maternal and neonatal adipose tissues. Mobilization of DDE from its storage sites is postulated to occur via its association with mobilized fatty acids and lipoproteins. The results provide an overall framework for evaluating the tissue dosimetry of DDE and for understanding how maternal exposure to DDE could affect perinatal sexual development in utero or in the early postnatal period.     

The metabolism of 14C-DDT, 14C-DDD, 14C-DDE and 14C-DDMU in rats and Japanese quail

The metabolism and excretion of 14C-DDT, 14C-DDD, 14C-DDE and 14C-DDMU were compared in male rats and male Japanese quail after i.p. injection. The rate of excretion of radioactivity was greater in the rat than in the quail for all compounds except DDMU. Skin and fat were major sites of residual radioactivity in both species, the compound administered and DDE accounting for most of the radioactivity except in the case of DDMU. The four radiolabelled compounds were all present in significant quantities in excreta of both species in unchanged forms. The metabolic patterns for DDT and DDD were similar in rat and quail, except that the rat formed 2,2-di(4-chlorophenyl)ethanol (DDOH) from DDT while the quail did not. Rat and quail metabolized DDE and DDMU differently. Ring-hydroxylated derivatives of DDE were formed only in the rat and analogous metabolites of DDMU were produced only by quail. Both species produced di(4-chlorophenyl)acetic acid as a metabolite of all four compounds administered, although the formation was generally less and slower in quail than rat. Comparative metabolism of DDMU with the other compounds indicated that this compound is not a metabolic intermediate in the metabolism of DDT in either rat or quail.     

Lymphatic transport and catabolism of therapeutic proteins after subcutaneous administration to rats and dogs

The mechanism underlying subcutaneous absorption of macromolecules and factors that can influence this process were studied in rats using PEGylated erythropoietins (EPOs) as model compounds. Using a thoracic lymph duct cannulation (LDC) model, we showed that PEGylated EPO was absorbed from the subcutaneous injection site mainly via the lymphatic system in rats, which is similar to previous reports in sheep. After subcutaneous administration, the serum exposure was reduced by ～70% in LDC animals compared with that in the control animals, and most of the systemically available dose was recovered in the lymph. In both LDC and intact rats, the total radioactivity recoveries in excreta after subcutaneous administration were high (70-80%), indicating that catabolism, not poor absorption, was the main cause for the observed low bioavailability (30-40%). Moreover, catabolism of PEGylated EPO was found with both rat subcutaneous tissue homogenate and lymph node cell suspensions, and a significant amount of dose-related breakdown fragments was found in the lymph of LDC rats. In addition, the bioavailability of PEGylated EPOs was shown to be 2- to 4-fold lower in "fat rats," indicating that physiologic features pertinent to lymphatic transport can have a profound impact on subcutaneous absorption. Limited studies in dogs also suggested similar subcutaneous absorption mechanisms. Collectively, our results suggest that the lymphatic absorption mechanism for macromolecules is probably conserved among commonly used preclinical species, e.g., rats and dogs, and that mechanistic understanding of the subcutaneous absorption mechanism and associated determinants should be helpful in biologic drug discovery and development.     

Effects of two isomers of DDT and their metabolite DDE on CYP1A1 and AhR function in human placental cells

The aim of this study was to investigate the actions of two isomers of DDT (p,p'-DDT, o,p'-DDT) and DDE (p,p'-DDE, o,p'-DDE) on the human placenta. We studied the effects of DDT and its metabolite DDE on CYP1A1 activity and on CYP1A1 and aryl hydrocarbon receptor (AhR) protein expression in placental cells. We used explants from third-trimester human placental tissue and JEG-3 cells, which are first-trimester human placenta cells. The main finding of this study was that the activity of CYP1A1 in the human placenta, measured in terms of ethoxyresorufin-O-deethylase (EROD) activity, was suppressed by treatment of 1, 10, and 100 ng/ml p,p'-DDT, o,p'-DDT, p,p'-DDE and o,p'-DDE. Immunoblot analyses indicated that both isomers of DDT and DDE inhibited the expression of CYP1A1 most effectively at 48 h and/or 72 h after the treatment. Because CYP1A1 activity is mediated by AhR, we evaluated the expression of AhR in placental tissue exposed to DDT and DDE for 1 h to 72 h. Our data showed that DDT and DDE gradually decreased the level of AhR protein, starting at 3 h or 24 h after the start of the experiment. Our results strongly support the involvement of the AhR/CYP1A1 signaling pathway in the mechanism of action of DDT and DDE in the human placenta.     

The short- and long-term effects of two isomers of DDT and their metabolite DDE on hormone secretion and survival of human choriocarcinoma JEG-3 cells

JEG-3 cells were used to compare the effects of two isomers of DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane), p,p'-DDT and o,p'-DDT and their metabolite DDE (1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene) on progesterone (P4) and human chorionic gonadotropin (hCG) secretion and cell apoptosis. Cells were treated with 1, 10, 100 ng/ml or 1 mug/ml of each compound for 24 or 72 h. Twenty four hours of exposure at 1 mug/ml of p,p'-DDT and o,p'-DDT decreased, whereas both DDEs, at all investigated concentrations, increased P4 secretion. Seventy two-hour exposure to all concentrations of both isomers of DDT and their metabolite DDE stimulated progesterone secretion. Statistically significant decrease in hCG secretion after 24 h and increase in hCG secretion after 72 h exposure for all investigated compounds was noted. Decrease in caspase-3 activity was observed in cells exposed to both isomers of DDT and its metabolites. These findings indicate that both isomers of DDT and their metabolite DDE are able to alter main placental hormone production and survival of JEG-3 cells in the concentration- and time-dependent manner.     

Bioaccumulation of DDT pesticide in cultured Asian seabass following dietary exposure

Bioaccumulation and metabolism of p,p'-DDT was studied in the marine carnivorous fish Lates calcarifer, Asian seabass, in a controlled aquaculture experiment. Over a 42-d period, seabass were fed pellets dosed with p,p'-DDT at environmentally realistic levels. Virtually all p,p'-DDT in pellets bioaccumulated in the fish with an uptake efficiency of 98%. The levels of p,p'-DDT and the metabolites p,p'-DDD and p,p'-DDE were analyzed in muscle, liver, visceral fat, brain, and remaining tissues. Partitioning of p,p'-DDT and its metabolites among the control, low-dose, and high-dose exposed seabass were 14.8% in muscle, 3.5% in liver, 37.1% in visceral fat, 0.11% in brain, and 45.5% in remaining tissues, where partitioning between tissues was a function of tissue lipid content. p,p'-DDT bioaccumulation increased linearly with exposure in visceral fat and muscle tissue. The metabolism of p,p'-DDT, which occurs mainly in the liver, resulted in the degradation of 2.5% of p,p'-DDT into p,p'-DDD. These new findings show that bioaccumulation processes at environmentally realistic ingestion exposure levels (ng/g) differ from previous DDT ingestion studies conducted at unrealistically high DDT levels (microg/g), highlighting the need to revise models on the transfer of persistent organic pollutants in the marine environment and aquaculture systems.     

The metabolism of 14C-DDT, 14C-DDD, 14C-DDE and 14C-DDMU in rats and Japanese quail

Abstract

1. The metabolism and excretion of ¹⁴C-DDT, ¹⁴C-DDD, ¹⁴C-DDE and ¹⁴C-DDMU were compared in male rats and male Japanese quail after i.p. injection.

2. The rate of excretion of radioactivity was greater in the rat than in the quail for all compounds except DDMU. Skin and fat were major sites of residual radioactivity in both species, the compound administered and DDE accounting for most of the radioactivity except in the case of DDMU.

3. The four radiolabelled compounds were all present in significant quantities in excreta of both species in unchanged forms.

4. The metabolic patterns for DDT and DDD were similar in rat and quail, except that the rat formed 2,2-di(4-chlorophenyl)ethanol (DDOH) from DDT while the quail did not.

5. Rat and quail metabolized DDE and DDMU differently. Ring-hydroxylated derivatives of DDE were formed only in the rat and analogous metabolites of DDMU were produced only by quail.

6. Both species produced di(4-chlorophenyl)acetic acid as a metabolite of all four compounds administered, although the formation was generally less and slower in quail than rat.

7. Comparative metabolism of DDMU with the other compounds indicated that this compound is not a metabolic intermediate in the metabolism of DDT in either rat or quail.     

Plant drugs with residues of organochlorine compounds. 2. Identification of residues of DDT and its analogs by comparison of gas chromatography on packed and capillary columns and GC/MS coupling

For the GC analysis of DDT isomers and metabolites in extracts of Flores Chamomillae end Radix Valerianae the separation on a packed QF-1/OV-17 column was compared with various capillary columns of the CP-Sil type. Identification of the individual compounds could be achieved by comparing the retention behavior, chemical transformation of DDT and DDE, as well as by capillary GC-MS using single ion monitoring of substance-characteristic ion mass. In this way, residues of p,p'-DDT, o,p'-DDE and p,p'-TDE could be identified.     

Sexual behavior of intact female rats after treatment with o,p'-DDT or p,p'-DDT

Sexual behavior of adult, female rats was tested following treatment with o,p'-DDT or with p,p'-DDT during either diestrus or proestrus. Both chlorinated compounds decreased lordosis behavior, but o,p'-DDT did so only after treatment on diestrus. p,p'-DDT in contrast, decreased sexual behavior under all treatment conditions. o,p'-DDT may have altered behavior by disrupting the estrous cycle while p,p'-DDT had a major effect on the female's proceptivity and receptivity without modifying vaginal cyclicity. p,p'-DDT disrupted sexual behavior at doses as low as 25 mg/kg while 100 to 200 mg/kg o,p'-DDT were required. Since commercially prepared DDT contained a predominant proportion of p,p'-DDT, these results suggest that many reproductive effects of DDT may have resulted from p,p'-DDT rather than from o,p'-DDT.     

Effect of lipid vehicle on the intestinal lymphatic transport of isotretinoin in the rat

Many lipophilic compounds are absorbed to some degree via the lymphatics, however, the mechanisms and factors controlling this absorption process are unclear. In order to provide some information on this area we have studied the effect of lipid vehicle on the lymphatic transport of isotretinoin following oral dosing to the rat. Oils containing higher percentages of the linoleate triglyceride ester appeared to promote both enhanced lymph flow and chylomicron concentration. Long chain triglyceride oils proved to be the most effective vehicles for increasing lymphatic transport - especially cottonseed oil and peanut oil. The solubility of the drug in the oil was also shown to be a key factor in lymphatic transport     

Uptake of lipophilic drugs by plasma derived isolated chylomicrons: Linear correlation with intestinal lymphatic bioavailability

Association of a drug with chylomicrons in the enterocyte is an essential step in the lymphatic absorption pathway. In this article, the uptake of lipophilic compounds by chylomicrons ex vivo was compared to the corresponding intestinal lymphatic bioavailability reported in rats in order to elucidate the degree of correlation and to evaluate the utilization of this correlation as a predictive measurement of the lymphatic bioavailability potential of lipophilic drugs. Nine lipophilic compounds (Vitamin D₃, Vitamin E, halofantrine, probucol, diazepam, testosterone, cyclosporin A, benzo[a]pyrene and p,p′-DDT) at a concentration of 1.75 × 10⁻⁶ M were incubated for 1 h with chylomicron emulsion separated from rat blood. A strong linear correlation was found between the degree of association of compounds with chylomicrons ex vivo and the lymphatic transport reported in rats (r² = 0.94, P < 0.0001), whereas log P and solubility in long chain triglycerides showed only moderate correlation with lymphatic bioavailability. The linear correlation between the degree of uptake of compounds by isolated chylomicrons and intestinal lymphatic transport suggests that the two processes are governed by similar factors. Thus, the degree of association of lipophilic compounds with isolated chylomicrons can be used as a simple screening model for estimation of intestinal lymphatic transport potential of drug molecules. This approach is important in view of the practical difficulties in direct determination of the lymphatic bioavailability in vivo.     

Lymphatic Transport of Liposome-Encapsulated Drugs Following Intraperitoneal Administration – Effect of Lipid Composition

Tumor cells often metastasize through lymphatic channels. It follows that localization of antitumor agents in the lymphatics may be therapeutically beneficial. This study determines the extent to which lipid composition controls lymphatic transport of a model compound (¹⁴C-sucrose) in liposomes following intraperitoneal administration in rats. All liposomes tested had mean diameters of approximately 0.2 µm. Liposomes were administerd to thoracic duct cannulated rats, and ¹⁴C was quantified in thoracic lymph, several lymph nodes, blood, urine, and peritoneal wash. Changing liposome composition altered the rate of absorption of ¹⁴C from the peritoneal cavity, stability in biological fluids, and the relative ability of liposomes to be retained by lymph nodes. Stability in biological fluids (plasma and lymph) appeared to be a reasonable predictor of observed lymph node recovery. Direct measures of lymph node level alone were poor measures of the ability of liposomes to function as prototypal lymphatic drug carriers. Neutral liposomes were better at reaching the general circulation following absorption from the peritoneal cavity.     

Dose-dependent excretion of DDE(1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) in rats

Dose-dependent excretion of p,pDDE in rats was investigated. p,pDDE itself was the major excreta in rats. But some o,p'isomer of DDE was detected in feces by GC-MS analysis. The excretion of p,p'DDE after a single administration was modified by its dose level.The time pattern of p,pDDE excretion agrees well with the modified Hill equation. The value of the equilibrium constant (K) increases in proportion to time t after p,pDDE administration.Using the modified Hill equation and the linear K equation, the excretion rate of p,pDDE during the experimental time t can be estimated. The estimated p,pDDE excretion rate in feces agrees well with the measurements.     

Solid lipid nanoparticles loading candesartan cilexetil enhance oral bioavailability: in vitro characteristics and absorption mechanism in rats

Candesartan cilexetil (CC) is widely used for the treatment of hypertension and heart failure, but it shows very poor aqueous solubility and very low oral absorption. In this work, CC-loaded solid lipid nanoparticles (CLNs) were successfully developed to improve the oral bioavailability. The physicochemical properties of CLNs were characterized, and the pharmacokinetic behavior of CLNs was evaluated in rats. CLNs exhibited nanometer-sized spherical particles with high entrapment efficiency (91.33%). The absorption of CLNs in the stomach was only 2.8% of that in intestine. Moreover, CLNs could be internalized into the enterocytes and then transported into the systemic circulation via the portal circulation and intestinal lymphatic pathway. The pharmacokinetic results indicated that the oral bioavailability of candesartan was obviously improved over 12-fold after incorporation into solid lipid nanoparticles. These results demonstrated that solid lipid nanoparticles have great potential for increasing oral bioavailability of lipophilic drugs such as CC. FROM THE CLINICAL EDITOR: Candesartan cilexetil is a potent angiotensin receptor inhibitor with low bioavailability due to poor aqueous solubility. In this work, solid lipid nanoparticles were used to improve the oral bioavailability 12-fold compared to standard preparation in rats, suggesting that a similar approach might be effective in future human applications.     

Effect of single and repeated in vitro exposure of ovarian follicles to o,p'-DDT and p,p'-DDT and their metabolites

The aim of the presented study was to compare the effect of o,p'-DDT [1,1-dichloro-2,2-bis-(p,p'-chlorophenyl)-ethylene] and p,p'-DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane] and their metabolites DDE and DDD on estradiol secretion by ovarian follicles, the target organs of environmental estrogens. Theca interna (Tc) and granulosa cells (Gc) were collected from medium size porcine follicles and cultured as a monolayer. The cells were initially cultured for 24 h to allow attachment to the plates and then media were changed for the new ones and o,p'-DDT and p,p'-DDT and their metabolites: o,p'-DDE, p,p'-DDE and o,p'-DDD were added at doses of 4, 40, 400 ng and 4 microg/ml medium to investigate dose-dependent effects. Media were collected after 24 h and frozen for estradiol content determination. When the effect of single and repeated exposure was investigated, the lowest dose of 4 ng/ml and the highest one of 4 microg/ml were chosen on the basis of the results of Experiment 1. o,p'-DDT exerted antiestrogenic action at all doses used while its metabolites and p,p'-DDT and its metabolites decreased estradiol secretion only when present in the medium at a dose of 4 ng/ml. The highest doses caused the increase in estradiol secretion. Parent o,p'-DDT and its metabolites showed antiestrogenic action after single exposure to 4 ng/ml while parent p,p'-DDT and its metabolites caused estrogenic action. All investigated compounds, except o,p'-DDT, increased estradiol secretion after single exposure to the dose of 4 microg/ml. Repeated exposure resulted in a massive antiestrogenic action of all investigated chemicals. In conclusion, our study points to time-dependent effect of DDT and its metabolites on ovarian follicles with the strongest estrogenic properties observed after single exposure and antiestrogenic action caused by repeated exposure. Given the duration of folliculogenesis, one can imagine many different potential mechanisms by which DDT could influence steroidogenesis.     

Organochlorine compounds in adipose tissue of Greenlanders and southern Danes.

Abdominal fat tissue samples from the general population of Greenland and from southern Denmark were analyzed for o,p'-DDE and p,p'-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], p,p,-DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane], o,p'-DDT and p,p'-DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], lindane (1,2,3,4,5,6-hexachlorocyclohexane), aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-endo-exo-1,4:5,8-dimethanonaphthalene), dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-endo-exo-1,4:5,8-dimethanonaphthalene), heptachlor (1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene), heptachlor epoxide, and polychlorinated biphenyls (PCBs). Fat tissue from Greenlanders contained significantly higher amounts of p,p'-DDE (p less than or equal to 0.05), p,p'-DDT (p less than or equal to 0.01), and total DDT (SIGMA DDT, the sum of DDT and its metabolites) (p less than or equal to 0.01) than southern Danes. Lindane, aldrin-like residue, dieldrin heptachlor-like residue, heptachlor epoxide, and PCBs were present in adipose tissue of both groups and there were no significant differences between the groups. The p,p'-DDE level in Greenland was lower than that in the United States, eastern Europe, and India. Among Greenlanders of different ages the highest sigma DDT was found in the age group of 22-45 yr; the content of PCBs increased with age. Among southern Danes the highest sigma DDT was found in a higher age group than in Greenlanders. Among Greenlanders the content of aldrin-like residue decreased with age and that of dieldrin increased with age. A significant correlation was found (on the basis of wet weight) between p,p'-DDE and PCB content in southern Danes (p less than or equal to 0.02). The correlation between sigma DDT and PCB content was also significant in this population (p less than or equal to 0.01). These two relationships were not significantly correlated for the Greenlanders. The DDE/PCB and sigma DDT/PCB ratios were higher in Greenlanders than in southern Danes. These ratios are age-dependent and are highest in the age group 22-45 yr among the Greenlanders. A low DDE/PCB ratio and a low sigma DDT/PCB ratio have been proposed as markers for industrialization. In Greenlanders p,p'-DDE represented about 70% of sigma DDT. For southern Danes this level was about 90%. The data obtained are presented and discussed on the basis of both lipid and wet weight levels.     

Effect of bile on absorption of mepitiostane by the lymphatic system in rats.

The effects of bile and site of gastrointestinal absorption on the lymphatic absorption of the highly lipophilic drug, mepitiostane were examined using thoracic duct-cannulated rats. The lymphatic absorption from the small intestine was very small in the absence of bile compared with that when bile was present. The lymphatic absorption was greatest when drug was administered to the upper small intestine with bile, was smaller for the lower regions of the small intestine, and was negligible for the stomach and the large intestine. A correlation was observed between the extent of lymphatic absorption and the secretion of chylomicron and very low density lipoproteins after administration to various regions with or without bile. The portal absorption data of mepitiostane confirmed that site specificity occurs in the partition of drug between blood and lymph.     

Organochlorine pesticides in human milk from different areas of Kenya 1983-1985

Residue levels of the chlorinated hydrocarbons p,p'-DDT (2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane), p,p'-DDE (2,2-bis(p-chlorophenyl)-1,1-dichloroethane), hexachlorobenzene (HCB), alpha-, beta-, and gamma-hexachlorocyclohexane (HCH), aldrin, dieldrin, and polychlorinated biphenyls (PCBs) were determined in human milk of Kenyan mothers living in different areas of Kenya. The main organochlorine contaminants found in all the milk samples analyzed were p,p'-DDT and p,p'-DDE. Great regional differences were found, and mean levels of sum DDT and DDT/DDE ratio ranged from 1.1 to 18.7 mg/kg milk fat and from 0.7 to 5.7, respectively. In general, relatively low residue levels of HCB, alpha-HCH, beta-HCH, aldrin, and dieldrin were detected in 59, 37, 27, 37, and 19%, respectively, of all the milk samples analyzed. Quantifiable residue levels of PCBs and alpha-HCH were not found. The results were examined in relation to differences in living conditions with regard to agricultural activities, dietary habits, and reported use of pesticides in the various sampling areas.     

Application of oral bioavailability surrogates in the design of orally active inhibitors of rhinovirus replication.

Previous studies in rats and humans demonstrated poor oral bioavailability of potent in vitro 2-aminobenzimidazole inhibitors of rhinovirus replication due to significant first-pass elimination and possibly also to poor aqueous solubility. Estimations of aqueous solubility, as well as measurements of caco-2 permeability and NADPH dependent compound loss in rat liver microsomal incubations were employed alongside traditional in vivo experiments in rats to guide subsequent chemistry efforts. Retention of activity upon replacement of the metabolically labile vinyl oxime in the lead molecule with a vinyl carboxamide was a major breakthrough; however, oral bioavailability among the latter compounds was variable. Based on the ability to independently measure solubility, permeability, and metabolic stability of new compounds, variable solubility across the series (ranging from approximately 1 to 10 microg/mL) was identified as the cause of the inconsistent performance. Subsequent efforts to improve solubility led to the discovery of highly soluble (>10 mg/mL) and potent dessulfonyl vinyl carboxamide benzimidazoles. Determination of the metabolic stability of these compounds as a surrogate of the extent of their first-pass elimination supported a prediction of excellent oral bioavailability. In comparison to the sulfonyl-containing vinyl carboxamides, caco-2 permeabilities were reduced 5 to 10-fold; however, these were considered to be in the range of well-absorbed compounds based on comparison to a series of reference compounds of known percentage absorption in humans. Subsequent experiments in the rat verified the oral bioavailability of these N-alkyl compounds, with one compound (368177) having an absolute oral bioavailability of 89.4%. The application of solubility and caco-2 permeability as surrogates for oral absorption potential, in conjunction with the use of microsomal incubations as a surrogate for first-pass metabolism, was shown to augment a rational chemistry approach to discover orally bioavailable inhibitors of rhinovirus replication. Future expanded use of these surrogates is planned.