Jean-Paul Payan

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.

Relationship Between Urinary Levels of 1-Hydroxypyrene and 3-Hydroxybenzo[ a ]pyrene for Workers Exposed to Polycyclic Aromatic Hydrocarbons

Biomonitoring of workers was carried out in three working areas--an artificial target factory, an aluminum plant, and an electrometallurgy plant--to assess exposure to PAHs. All the 48 hr-voided urine samples of the exposed workers were collected and the 3-hydroxybenzo[ a ]pyrene (3-OHB a P) and 1-hydroxypyrene (1-OHPy) analyzed according to procedures using automated column-switching high-performance liquid chromatography. The exposure profiles indicate an important lag between the excretion of the two metabolites: the maximum of 3-OHB a P urinary excretion is observed 10 to 17 hr after the 1-OHPy maximum, with a much closer correlation ( r = 0.81) than that obtained with both metabolites in real time ( r = 0.21). This delayed excretion agrees with data from animal intoxication studies (intravenous administration or inhalation). Mean ratios of 1-OHPy to 3-OHB a P were studied without lag (varying from 2,230 to 15,330) and with a lag of 16 hr (varying from 940 to 8,390).

Comparison of percutaneous absorption and metabolism of di-n-butylphthalate in various species.

An ex vivo study of the percutaneous absorption of di-n-butylphthalate (DBP) showed that DBP was completely hydrolysed by esterases during penetration through rat skin. Fluxes were dependent on the esterase activity in the skin. The aim of this study was to determine the nature of the esterases involved in the hydrolysis of DBP in the skin. The relation between the percutaneous absorption of DBP and the epidermis/dermis esterase activity was determined in human, rat, rabbit, guinea-pig and mouse skin. An animal model was tested to estimate the human percutaneous absorption of lipophilic ester substances such as DBP. The nature of the esterases was determined by inhibition study in epidermis and dermis homogenates. A topical application of neat [(14)C]-DBP was used to determine ex vivo fluxes. Monobutylphthalate (MBP) levels in each skin layer were determined by high-performance-liquid-chromatography (HPLC). DBP was mainly hydrolysed by skin carboxyesterases for the all studied species. Unlike MBP levels in the skin, epidermis or whole skin esterase activity was not related to the DBP fluxes (hairless rat>hairy rat>hairless mouse=rabbit>guinea-pig>human) of all studied species. Therefore prediction of the results in the human being by extrapolation from animal data should be done carefully.

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.

Modulation of acrylonitrile-induced embryotoxicity in vitro by glutathione depletion

The effects of glutathione (GSH) depletion on the embryotoxicity of acrylonitrile were assessed in vitro using the rat whole-embryo culture system. Day 10 rat embryos were cultured in rat serum medium for 6 h in the presence of 250 μMl-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of GSH synthesis, to deplete GSH in both embryo and visceral yolk sac. Following pretreatment, conceptuses were cultured for an additional 21 h in the presence of 152, 228, or 304 μM acrylonitrile. At the end of the culture period, conceptuses were assessed for survival, growth and development, malformations, and the protein and glutathione content of embryos and yolk sacs were assayed. Acrylonitrile alone produced concentrationrelated and statistically significant decreases in yolk sac diameter, crown-rump length, head length and number of somite pairs, as well as in embryonic and yolk sac proteins. The chemical also caused dysmorphogenesis of the brain and of the caudal extremity, and a concentration-related and statistically significant increase in GSH content in the yolk sac. Pretreatment with BSO significantly enhanced the embryotoxic effects of acrylonitrile. The conceptuses displayed further decreases in functional yolk sac circulation, yolk sac diameter, crown-rump and head length, when compared to either acrylonitrile or BSO alone. The incidence of caudal malformations and the severity of brain malformations produced by acrylonitrile were also increased. Marked decreases in embryonic and yolk sac GSH contents were observed after exposure to BSO alone or in combination with acrylonitrile. Thus, depletion in embryonic and yolk sac GSH by BSO enhanced teratogenic and growth retarding effects of acrylonitrile in vitro, suggesting that GSH plays a critical role in modulating acrylonitrileelicited embryotoxicity.

Assessment of urinary rat β2-microglobulin by enzyme immunoassay

Abstract An enzyme immunoassay (EIA) was developed using unlabelled and peroxidase-labelled rabbit antibodies to assess urinary rat β 2 -microglobulin (β 2 -m) excretion. It consisted in the adsorption of rabbit anti-rat β 2 -m immunoglobulin to a polystyrene surface, the addition of β 2 -m samples or standard and the addition of peroxidase-labelled rabbit anti-rat β 2 -m immunoglobulin. After addition of hydrogen peroxide and o -phenylenediamine, the enzyme activity of the solid phase was measured photometrically at 490 nm. Analytical recovery of pure β 2 -m in urine was 1029%. From determinations made on normal female and male rats, the ranges of 24-h urine β 2 -m individually excreted were found to be 0.84–4.77 and 3.7–17.3 μg, respectively. The means ± SEM were 2.49 ± 0.17 and 10.2 ± 0.55 μg. EIA was of value in evidencing the tubule-damaging properties of sodium chromate and hexachloro-1,3-butadiene in rats.

Pregnancy-associated changes in renal toxicity of cadmium-metallothionein: possible role of intracellular metallothionein

Nulliparous female, 10-day and 20-day pregnant rats were injected intraperitoneally with saline or labelled cadmium-metallothionein (109Cd-MTh) at a single dose of 25 or 250 micrograms Cd as cadmium-metallothionein (Cd-MTh)/kg and sacrificed at 24 h. The renal toxicity was manifested by increased 24-h urinary excretion of beta 2-microglobulin (beta 2-m) and the increased number of damaged convoluted proximal tubules at 24 h. The renal excretion of 109Cd and 109Cd content in the maternal liver and kidney and in the foeto-placental unit were determined. The binding of 109Cd to kidney proteins and the level of intracellular metallothionein (MTh) in livers and kidneys were also determined. It was found that the nephrotoxicity of injected Cd-MTh did not differ in nulliparous and 10-day pregnant rats. This result was consistent with the absence of difference in the renal uptake of 109Cd, its binding to kidney proteins and in the content of endogenous MTh in the kidneys between nulliparous and 10-day pregnant rats. In contrast, 20-day pregnant rats exhibited much more nephrotoxicity than nulliparous rats. The most prominent finding in relation to the extreme sensitivity of 20-day pregnant rats was a lower basal level of intracellular MTh in the kidneys and the accumulation of 109Cd in the high molecular weight proteins in the soluble fraction. It is suggested that the decrease of intracellular MTh in the kidneys of 20-day pregnant rats is the reason for the low protection against the renal toxicity of injected Cd-MTh.

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.

A sensitive cadmium-affinity assay for the determination of thionein in urine

A sensitive cadmium-affinity assay was developed for measurement of urinary thionein (Th) level. Acidification with HCl (2.5 M) was used to remove metal ions from purified urinary metallothionein (MTh), adsorbed on activated polystyrene tubes. It was found that the amount of 109Cd bound to standard Th (rabbit Th) was proportional to that of Th in the concentration range of 20-6000 ng/ml. The method exhibited a sensitivity below 20 ng/ml and a precision of about 5%. The results of the Cd-affinity assay were unaffected by dilution of, or addition of standard Th to urine samples. Under the latter circumstances, the Cd-affinity assay was performed with a mean analytical recovery of 100.3 +/- 4%. Addition of Cd, Zn, Hg and Cu (50 micrograms each) or glutathione and cysteine (0.1 mmol) to urine specimens (1 ml) did not interfere with the determination of Th. The mean values of urinary Th in healthy subjects were 200 +/- 53 micrograms/g creatinine (n = 9) and 256 +/- 97 micrograms/g creatinine (n = 8) for men and women respectively. The mean daily excretions of Th by non-fasting female and male healthy adult rats were 9.95 +/- 2.7 micrograms (n = 10) and 18.2 +/- 3.9 micrograms, respectively. The Cd-affinity assay succeeded in indicating Cd exposure and/or development of Cd toxicity in rats.