In vivo and in vitro percutaneous absorption of [(14)C]di-N-butylphthalate in rat.

This study evaluated the toxicokinetics of [(14)C]di-n-butylphthalate ([(14)C]DBP) after an intravenous administration (1 and 10 mg/kg, in Cremophor) or a topical application (10 microl/cm(2); 10 cm(2), neat) in haired male Sprague-Dawley rats. Additional in vivo and in vitro percutaneous penetration studies of [(14)C]DBP were conducted on male and female haired rats and male hairless rats. After intravenous administration, unchanged DBP disappeared rapidly from the plasma, following a two-exponential function (T1/2beta = 5-7 min). The peak levels of monobutylphthalate (MBP) and its glucuronide conjugate (MBP-Gluc) occurred 1 to 2 and 20 to 30 min after administration, respectively. These metabolites were intensively and rapidly excreted in urine (57% of the dose). However, about 35% of the dose recovered in urine was primarily excreted in bile (mainly as MBP-Gluc) and underwent hepatobiliary recycling. Unchanged DBP was barely detectable in excreta. DBP rapidly penetrated the skin, which constituted a reservoir. The absorption flux determined for 0.5 to 8 and 8 to 48 h of exposure were 43 and 156 microg/cm(2)/h, respectively. The higher flux may be due to radial diffusion of DBP in the stratum and/or epidermis. The in vivo and in vitro experiments revealed that DBP was intensively metabolized into the skin. In vivo percutaneous absorption flux was very similar in male and female haired rats. In contrast, the percutaneous absorption determined in vivo and in vitro was higher in hairless than in haired male rats. Absorption flux was accurately estimated from urinary excretion rate of MBP or MBP-Gluc.

Toxicokinetics and metabolism of 1,2-diethylbenzene in male Sprague Dawley rats--part 2: evidence for in vitro and in vivo stereoselectivity of 1,2-diethylbenzene metabolism.

In a previous study, it was shown that the neurotoxic compound 1,2-diethylbenzene (1,2-DEB) is mainly hydroxylated in the alkyl chain to give 1-(2'-ethylphenyl)ethanol (1,2-EPE) and excreted in urine of rats as two glucuronide compounds (GA1 and GA2). Some findings have suggested that the two enantiomers of 1,2-EPE are formed in vivo. In the present study, a chiral high-performance liquid chromatography method was developed to separate the two enantiomers of 1,2-EPE from a synthesized racemic mixture. Absolute configuration of both enantiomers was determined after esterification with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid and analysis of their (1)H NMR spectra in CCl(4) added with Eu (fod)(3). The two main urinary metabolites, GA1 and GA2, from [(14)C]1,2-DEB-treated Sprague-Dawley rats (80 mg/kg, i.p.) were identified, after hydrolysis with beta-glucuronidase from Escherichia coli, as (R) and (S) glucuronide conjugates of 1,2-EPE, respectively. In vitro hydroxylation of 1,2-DEB and glucuroconjugation of 1,2-EPE were under stereoselective control in S9 fraction or microsomes from male Sprague-Dawley rat liver. The V(max) and K(m) constants for (R)1,2-EPE enantiomer formation determined in S9 fraction were greater than those for the (S) enantiomer. In the plasma of bile duct-cannulated rats, the ratio was 1.2 +/- 0.02 over the 1- to 4-h period after oral administration of [(14)C]1,2-DEB (100 mg/kg). In contrast, the glucuroconjugation rate of (S)1,2-DEB enantiomer was 4 times that of (R)1,2-EPE glucuroconjugation. A similar ratio of (R) to (S)1,2-EPE glucuronide conjugates was obtained in the plasma of bile duct-cannulated rats.

Assessment of the developmental toxicity and placental transfer of 1,2-diethylbenzene in rats.

Sprague-Dawley rats were administered 1,2-diethylbenzene (1,2-DEB) by gavage on gestational days (GD) 6 through 20 at dose levels of 0 (corn oil), 5, 15, 25 or 35 mg/kg. The dams were euthanized on GD21 and the offspring were weighed and examined for external, visceral and skeletal alterations. Maternal toxicity, indicated by significant decreases in body weight gain and food consumption, was observed at doses of 15 mg/kg and above. Developmental toxicity, expressed as significantly reduced foetal body weights, was seen at doses of 15 mg/kg and higher. There was no evidence of embryolethal or teratogenic effects at any dose tested. The placental transfer of 1,2-DEB was examined after a single oral dose of 25 mg [14C]1,2-DEB/kg on GD18. Maternal and foetal tissues were collected at intervals from 1 to 48 hours. Placental and foetal tissues accounted for less than 0.35% of the administered dose. Levels of radiocarbon in foetuses were lower than those in maternal plasma and placenta at all time points. Analysis performed at 1, 2 and 4 hours indicated that ethyl acetate extractable (acidic) metabolites were predominant in the maternal plasma while n-hexane extractable (neutral) compounds represented the major part of radioactivity in the placenta and foetus. In conclusion, this study demonstrated that 1,2-DEB causes mild foetotoxicity at maternal toxic doses and that the exposure of the developing rat foetus to 1,2-DEB and/or metabolites after maternal administration of 1,2-DEB in late gestation is small.

Toxicokinetics of 1,2-diethylbenzene in male Sprague-Dawley rats-part 1: excretion and metabolism of [(14)C]1,2-diethylbenzene.

The excretion and metabolism of neurotoxic 1,2-diethylbenzene (1, 2-DEB) was studied in male Sprague-Dawley rats after i.v. (1 mg/kg) or oral (1 or 100 mg/kg) administration of 1,2-diethyl[U-(14)C]benzene ([(14)C]1,2-DEB). Whatever the treatment, radioactivity was mainly excreted in urine (65-76% of the dose) and to a lower extent in feces (15-23% of the dose), or via exhaled air (3-5% of the dose). However, experiments with rats fitted with a biliary cannula demonstrated that about 52 to 64% of the administered doses (1 or 100 mg/kg) were initially excreted in bile. Biliary metabolites were extensively reabsorbed from the gut and ultimately excreted in urine after several enterohepatic circulations. Insignificant amounts of unchanged 1,2-DEB were recovered in the different excreta (urine, bile, and feces). As reported previously, presence of 1-(2'-ethylphenyl)ethanol (EPE) was confirmed in urine and demonstrated in bile and feces. The two main [(14)C]1,2-DEB metabolites accounted for 57 to 79% of urinary and biliary radioactivity, respectively. Beta-Glucuronidase hydrolysis and electron impact mass spectra results strongly supported their glucuronide structure. Additionally, these two main metabolites were thought to be the glucuronide conjugates of the two potential enantiomers of EPE. The results indicate that the main initial conversion step of the primary metabolic pathway of 1,2-DEB appears to be the hydroxylation of the alpha-carbon atom of the side chain. The presence of two glucuronide conjugates of EPE in the urine in a ratio different from one suggests that the metabolic conversion of 1, 2-DEB is under stereochemical control.

Assessment of the developmental toxicity, metabolism, and placental transfer of Di-n-butyl phthalate administered to pregnant rats.

The developmental toxicity and placental transfer of di-n-butyl phthalate (DBP) were evaluated in Sprague-Dawley rats given a single oral dose of DBP on Gestational Day 14. In the developmental toxicity study, dams were dosed with 0, 0.5, 1, 1.5, or 2 g DBP/kg and were necropsied on GD21. Increased incidence of resorptions and reduced fetal body weight were observed at 1.5 and 2 g/kg. Higher incidences of skeletal variations were found at doses > or = at 1 g/kg. No embryotoxic or teratogenic effects were observed at a dose of 0.5 g/kg. In the placental transfer study, dams were dosed with 0.5 or 1.5 g [14C]DBP/kg. Maternal and embryonic tissues were collected at intervals from 0.5 to 48 h. Embryonic tissues accounted for less than 0.12-0.15% of the administered dose. Levels of radiocarbon in placenta and embryo were one-third or less of those in maternal plasma. No accumulation of radioactivity was observed in the maternal or embryonic tissues. From HPLC analyses, it was shown that unchanged DBP and its metabolites mono-n-butyl phthalate (MBP) and MBP glucuronide were rapidly transferred to the embryonic tissues, where their levels were constantly lower than those in maternal plasma. MBP accounted for most of the radioactivity recovered in maternal plasma, placenta, and embryo. Unchanged DBP was found only in small amounts. These findings support the hypothesis that MBP, a potent teratogen, largely contributes to the embryotoxic effects of DBP.

Assessment of the developmental toxicity, metabolism, and placental transfer of N,N-dimethylformamide administered to pregnant rats.

This study evaluates the developmental toxicity and placental and milk transfer of N,N-dimethylformamide (DMF) in rats. Sprague-Dawley rats were given 0, 50, 100, 200, and 300 mg DMF/kg/day, by gavage, on Gestational Days (GD) 6 through 20. Maternal toxicity was indicated by depressions in weight gain and food consumption at doses >/=100 mg/kg. Fetal toxicity was indicated by decreased fetal body weight at doses >/=100 mg/kg, and by increased incidences of two skeletal variations (absent or poorly ossified supraoccipital and sternebrae) at 200 and 300 mg/kg. Thus, the maternal and developmental no-observed-adverse-effect level was 50 mg/kg/day. The time course disposition of [14C]DMF was examined over a 48-hr period in GD12- and GD18-pregnant rats after a single oral dose of 100 mg [14C]DMF/kg. Peak concentrations of radiocarbon occurred within 1 hr after dosing. Embryonic (GD12) and fetal (GD18) tissues accounted for 0.15 and 6% of the administered dose, respectively. Levels of radiocarbon in embryonic and fetal tissues were equal or slightly less than in maternal plasma up to 8 and 24 hr, respectively, and higher thereafter. HPLC analysis performed at intervals from 1 to 8 hr on GD12 and 1-24 hr on GD18 indicated that unchanged DMF and metabolites were readily transferred to the embryonic and fetal tissues, where their levels were generally equal to those in maternal plasma. The parent compound accounted for most of the radioactivity until 4-8 hr and then decreased. N-Hydroxymethyl-N-methylformamide (HMMF) and N-methylformamide (NMF) were the predominent metabolites and increased with time. Much lower concentrations were found for formamide and N-acetyl-S-(N-methylcarbamoyl)cysteine. Transfer of radioactivity into milk was studied in dams given a single oral administration of 100 mg [14C]DMF on Lactation Day 14. DMF, HMMF, and NMF were found in the milk at concentrations equal to those in plasma.

Assessment of the developmental toxicity and placental transfer of 1,2-dichloroethane in rats.

This study evaluates the developmental toxicity and placental transfer of 1,2-dichloroethane (DCE) in rats. Sprague-Dawley rats were given 0-2.4 mmol DCE kg-1 day-1 by gavage, or were exposed for 6 hr per day to 0-300 ppm DCE by inhalation, from Day 6 to 20 of gestation. Maternal toxicity was observed after inhalation exposure to 300 ppm DCE and oral administration of 2.0 or 2.4 mmol DCE kg-1. There was no evidence of altered growth nor teratogenic effects after either inhalation or oral administration of DCE at any concentration tested. The time course disposition of 14C was examined over a 48-hr period in 12- and 18-day pregnant rats after a single oral dose of 1.6 mmol [14C]DCE kg-1. Peak concentrations of radiocarbon occurred between 2 and 4 hr postdose. Conceptus (Day 12) and fetal (Day 18) tissues accounted for 0.06 and 0.4% of the administered dose, respectively. Up to 4 hr, levels of radiocarbon in placenta and fetus were slightly less than in maternal plasma of 18-day pregnant rats and were two to five times higher at later periods. At 2 hr, unchanged DCE accounted for most of radioactivity (78-86%) recovered in maternal plasma, placenta, and fetus. Acidic metabolites and radioactivity bound to macromolecules increased up to 24 hr (0.01 mumol-eq DCE g-1) in either placental or fetal tissues. Thereafter, their levels declined more slowly than those in the maternal plasma. Results from this developmental toxicity study in rats confirm embryonic exposure to radiocarbon associated with [14C]DCE and/or its metabolites and has demonstrated the lack of observable teratogenic effects.

Specific amino acids modulate the embryotoxicity of nickel chloride and its transfer to the rat embryo in vitro.

To investigate the effects of amino acids on the embryotoxicity and placental transfer of nickel chloride (NiCl2), Day 10 rat embryos were cultured in rat serum medium containing NiCl2 or 63NiCl2 (0.34 or 0.68 mM Ni), with or without L-histidine (2 mM), L-aspartic acid, glycine (2 or 8 mM), or L-cysteine (2 mM). After 26 hr, conceptuses were assessed for survival, growth and development, and malformations. The 63Ni contents of embryos and yolk sacs and the extent of 63Ni binding to the proteins of the culture medium were also determined. NiCl2 alone did not affect the embryonic development at 0.34 mM and caused growth retardation and brain and caudal abnormalities at 0.68 mM. Coincubation of L-histidine with 0.34 mM Ni increased Ni concentrations in embryonic tissues compared to 0.34 mM 63Ni alone, but did not elicit NiCl2 embryotoxicity. Coincubation of L-cysteine with 0.34 mM Ni elicited growth retardation and brain abnormalities caused by NiCl2 and increased yolk sac concentrations of 63Ni compared to 0.34 mM 63Ni alone. In contrast, coincubation of L-histidine, L-cysteine, or L-aspartic acid with 0.68 mM Ni reduced the growth retardation and the incidence and/or severity of brain defects caused by NiCl2 and decreased the concentrations of 63Ni in the yolk sacs, compared to 0.68 mM 63Ni alone. L-Histidine also reduced the percentage of NiCl2-elicited caudal defects. Coincubation with glycine did not NiCl2-elicited caudal defects. Coincubation with glycine did not affect the embryotoxic profile, nor the placental transfer of NiCl2. In the presence of L-histidine, L-cysteine, or L-aspartic acid, there was a shift of 63Ni binding from the high-molecular-weight proteins of the culture medium to the low-molecular-weight fraction. Thus, specific extracellular amino acids can modulate the embryotoxicity and placental transfer of NiCl2 in vitro. The pattern of this modulation is dependent on the concentration of NiCl2, as well as on the amino acid.

Urinary thiodiglycolic acid and thioether excretion in male rats dosed with 1,2-dichloroethane.

1,2-dichloroethane (DCE) is extensively metabolized and partially excreted in urine as thioether compounds, which include thiodiglycolic acid (TDGA). In this study, we have compared the urinary excretion of TDGA and thioethers in the rat after administration of increasing doses of DCE. Male Sprague Dawley rats were given a single oral dose of labelled [14C]DCE (0.125 to 8.08 mmol kg-1 body wt.) and 24-h urine samples were collected. The TDGA and thioethers were determined in urine by a gas chromatography method and by the thioether assay after alkaline hydrolysis, respectively. The percentage of the administered radioactivity that was excreted in urine decreased with increasing dose of DCE and ranged between 63 and 7.4%. The amount of TDGA increased proportionally to the DCE dose up to 1.01 mmol DCE kg-1 body wt. and corresponded to 0.22 mmol TDGA mmol-1 DCE. Up to 0.25 mmol DCE kg-1 body wt., the amount of thioethers recovered in urine was not significantly different as compared to the vehicle control group (11.8 +/- 0.6 mumol SH equiv. kg-1 body wt., n = 10). From the 0.25-4.04 mmol DCE kg-1 body wt. dose, the amount of thioethers increased linearly with the dose of DCE and corresponded to 0.028 mmol SH equiv. mmol-1 DCE. The ratio between urinary thioethers and TDGA increased with the DCE dose and reached 0.17 +/- 0.01 (n = 5) at a dose of 8.08 mmol DCE kg-1 body wt. Moreover, TDGA contents determined in urine by gas chromatography before and after alkaline hydrolysis were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial contribution of biliary metabolites to nephrotoxicity, renal content and excretion of [14C]hexachloro-1,3-butadiene in rats.

Male Sprague Dawley rats with cannulated bile duct (BDC rats) received 100 or 200 mg kg-1 labelled hexachloro-1,3-butadiene ([14C]HCBD) by gavage 1 h (BDC1 rats) or 24 h (BDC24 rats) after surgical cannula implantation. Twenty-four hours after treatment with HCBD, rats were examined histochemically and biochemically for kidney damage. Urine, faeces, liver and kidney radioactivities were also measured in 24-h samples. Results were compared with those obtained from non-cannulated (NC) rats. Bile-duct cannulation did not completely protect against HCBD-induced kidney damage. The 24-h [14C] urinary excretion and tissue content was 30-50% lower in BDC rats compared to NC rats and correlated well with the toxicity findings. BDC1 rats appeared to be much more resistant to HCBD treatment than BDC24 rats. Since faecal [14C] radioactivity extractable by diethyl ether at neutral pH in BDC1 rats was twice that measured in BDC24 rats, the greater resistance was attributed to a higher deficiency in the gastrointestinal absorption of unchanged HCBD. The present results reveal that the biliary metabolites of HCBD are not solely responsible for kidney toxicity as previously assumed. They suggest a sinusoidal efflux of the HCBD conjugates from the liver.

Biliary excretion of hexachloro-1,3-butadiene and its relevance to tissue uptake and renal excretion in male rats.

Renal, biliary, pulmonary and faecal excretion experiments were carried out with labelled hexachloro-1,3-butadiene [( 14C]HCBD) in male Sprague-Dawley rats, given orally (p.o.) and intravenously (i.v.) in doses of 1 and 100 mg kg-1 as a solution in polyethylene glycol. The radioactivity excreted over 72 h was determined in rats fitted with exteriorized biliary cannulae and in rats whose bile ducts remained fully functional, respectively. In addition, bile duct-duodenum cannula-linked rats, of which the donor was given 100 mg kg-1 [14C]HCBD orally and the recipient had also a bile fistula, were examined within 30 h for radioactivity in the excreta, the kidney, the liver and the plasma. In non-cannulated rats, fractional urinary excretion decreased when the dosage increased and amounted to 23% and 8.6% after i.v. injection or 18.5% and 8.9% after p.o. administration of 1 and 100 mg kg-1, respectively. Pulmonary excretion of radioactivity was less than 9% and was not affected by the increase in dosage. In bile duct-cannulated rats, fractional urinary excretions were similar irrespective of the dose and the route of administration and amounted to ca. 7.5% of the dose. Decrease in fractional biliary excretion occurred with increase in dosage (88.7% vs 72%) after i.v. injection and (66.8% vs 58%) after gavage. In cannulated rats, faecal excretion was less than 0.5% after i.v. injection and accounted for 3% and 16% of the dose after p.o. administration of 1 and 100 mg kg-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)