Developmental and reproductive toxicity studies on artemisone.

BACKGROUND: In order to justify clinical studies in women of child-bearing age with artemisone, a new artimisinin derivative, studies to assess fertility and early embryonic development in rats, developmental toxicity in rats and rabbits, and peri-post natal development in rats were performed. METHODS AND RESULTS: In the study on fertility and early embryonic development (dose levels 0-5-20-80 mg/kg bw/day), doses inducing clinical and organ toxicity were used. Only in severe toxicity conditions, a reduction of the number of estruses, a prolonged time to insemination, decreased numbers of corpora lutea, implantation sites, and viable fetuses were found. Two developmental toxicity studies were performed in rats (dose levels 0-1-2 mg/kg bw/day) and rabbits (dose levels 0-2.5-5.0-7.5 mg/kg bw/day). It was shown that rats were about 5 times more sensitive than rabbits. In rats, artemisone induced total litter loss (late resorptions) at 2 mg/kg body weight and above with an increased incidence of a common vascular variation and retarded ossification at this dose. In rabbits, maternal toxicity, abortion and a slightly increased incidence of cardiac ventricular septal defects was observed at 7.5 mg/kg body weight. In a pre- and postnatal developmental toxicity study in rats (dose levels 0-1-2-4 mg/kg bw/day), 4 mg/kg body weight artemisone induced clinical symptoms and affected postnatal survival, body weight gain in the F1 pups, and motor activity. CONCLUSIONS: In summary, artemisone was shown to be embryo- and fetotoxic and induced cardiac ventricular septal defects and retarded ossification in dosages where total litter loss and abortions were observed. However, no effect on reproductive and developmental parameters below severe toxic dosages could be observed.

Evaluation of the rodent Hershberger bioassay: testing of coded chemicals and supplementary molecular-biological and biochemical investigations.

Under the auspices of the Organization for Economic Cooperation and Development (OECD) the Hershberger assay is being validated as an in vivo screen for compounds with (anti)androgenic potential. We participated in the final activity, the testing of coded chemicals. Test compounds included trenbolone (TREN; 1.5, 40 mg/kg), testosterone propionate (TP; 0.4 mg/kg), flutamide (FLUT; 3mg/kg), linuron (LIN; 10, 100mg/kg), 1,1-bis-(4-chlorophenyl)-2,2-dichloroethylene (p,p'-DDE; 16, 160 mg/kg), and two negative reference substances, i.e., compounds not considered to affect androgen-sensitive tissue weights (ASTWs) in the Hershberger assay, namely 4-nonylphenol (NP; 160 mg/kg) and 2,4-dinitrophenol (DNP; 10mg/kg); TREN, LIN, p,p'-DDE, NP, and DNP being used under code. Compounds were administered for 10 days by oral intubation or subcutaneous injection (TP). Additional investigations not mandatorily requested by OECD included organ gravimetry of the liver, gene expression analysis in prostate using quantitative RT PCR for prostate specific binding protein polypeptide C3 (PBPC3) and ornithine decarboxylase 1 (ODC1) and determination of testosterone metabolizing and phase II conjugating enzymes in the liver. After submission of all study reports to OECD by participants uncoding revealed the following results: (A) When assessing androgenic potential in castrated rats, administration of TREN increased the weights of ventral prostate (VP), seminal vesicles (SV), glans penis, levator ani and bulbocavernosus muscles, and Cowper's glands at the high dose. A similar or stronger (VP, SV) increase of ASTWs was observed for TP; NP and DNP were ineffective. TREN dose-dependently increased gene expression of ODC1 and PBPC3, TP induced expression of these genes even more strongly (almost) to the level of untreated intact animals, whereas NP and DNP were inactive. Liver enzyme activities depending on physiological androgen levels were lower in castrated than in intact rats and could not be restored by androgen treatment. (B) When assessing antiandrogenic potential in TP-supplemented castrated rats, administration of LIN and p,p'-DDE decreased ASTWs only at the high dose. FLUT even more effectively decreased ASTWs, NP and DNP were again without effect. Decreases in androgen-responsive gene expression in the prostate corresponding to the organ weight changes were only observed for p,p'-DDE (high dose) and flutamide (PBPC3 only). p,p'-DDE dose-dependently induced liver weights and most liver enzyme activities including androgen-dependent ones. Our study accurately reproduced ASTW changes obtained in previous studies also under code suggesting that the Hershberger assay is a robust tool to screen for an (anti)androgenic potential. Assessment of ODC1 and PBPC3 gene expression in prostate, however, may only represent a sensitive tool for the detection of an androgenic potential. Finally, p,p'-DDE may affect ASTWs by several mechanisms including enhanced testosterone metabolism.

Differential response of immature rat uterine tissue to ethinylestradiol and the red wine constituent resveratrol.

The stilbene derivative resveratrol (RES) is a phytoalexin of grapes, peanuts and other fruits. It is structurally related to stilbene estrogens and an estrogenic potential of RES has recently been demonstrated in a number of in vitro studies. In this investigation, the uterotrophic responses of immature Wistar rats to subcutaneous administration of RES (18, 58, and 575 mg/ kg) and the reference estrogen ethinylestradiol (EE2; 0.3, 1, 3, 30 microg/kg) on three consecutive days were determined. Uterine weight, histopathological changes, immunohistochemical expression of nuclear estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) protein, gene expression of ERalpha and PR at the messenger ribonucleic acid (mRNA) level and peroxidase induction were examined. EE2 dose dependently increased uterine weight, enlarged the uterine lumen and induced hypertrophy of epithelial, stromal and myometrial cells. Expression of ERalpha protein in epithelial, stromal and myometrial nuclei and of PR protein in epithelial nuclei was reduced in EE2-treated rats, while PR protein in stromal and myometrial nuclei was increased in a dose-dependent manner. EE2 increased messenger ribonucleic acid (mRNA) levels of uterine PR and induced peroxidase activity. In contrast, RES rather mildly decreased uterine weight, while histology did not reveal differences between controls and RES-treated rats. Expression of nuclear ERalpha protein was dose dependently decreased in epithelial, stromal and myometrial cells of RES-treated rats, while nuclear PR protein content was similar in controls and RES-treated rats. Following administration of RES, a trend toward reduced levels of ERalpha and PR mRNA was found, while no peroxidase induction occurred. Plasma levels of RES, 45 min after the administration of a single subcutaneous dose of 500 mg/kg, were in the range 1-2 microM. In summary, an estrogenic potential of RES could not be substantiated in this in vivo study, although the most effective route of administration and extremely high doses were used and plasma levels were in the range reported to be effective in vitro. Whether other pharmacological properties of RES could mediate the observed changes in RES-treated animals is discussed.

D-glucose combined chronic toxicity and carcinogenicity studies in Sprague-Dawley rats and Syrian golden hamsters.

After an initial period of 16 weeks with increasing concentrations, D-glucose was administered at 30% in the diet to 50 male and 50 female Sprague-Dawley rats from the 17th to the 112th study week. Additional 10 male and 10 female animals were treated for 14 months and then sacrificed for interim examination. Groups of 60 male and 60 female Syrian golden hamsters received D-glucose in the form of 20% solution in tap water for a period of 80 weeks. In each case, groups consisting of an equal number of untreated animals served as controls. General behavior and mortality were not affected by the treatment. The rats and hamsters treated with glucose showed significantly higher body weights of up to a maximum of 16% in male and 26% in female rats, or 15% in male and female hamsters. In rats, the increase was evident by week 14, and in the hamsters by week 10. Glucose-dosed rats displayed a slightly increased feed intake and a reduced water intake. Both parameters, however, were not influenced in hamsters. Hematological and histopathological examination showed no pertinent changes in hematopoetic tissue. Sharply increased blood glucose and renal glucose excretion values were present in rats beginning with 18 months and were indicative of the development of non-insulin-dependent diabetes mellitus (NIDDM). The insulin concentrations in peripheral blood were not appreciably affected, although there was a trend to higher values in males at all evaluation times and in females only at 3 months. Pathological evaluation did not show any compound related non-neoplastic lesions. The incidences of islet cell adenomas in the pancreas of male rats were significantly increased and the cortical adenomas in the adrenals of females were decreased. In addition, the mammary gland adenomas (in females) and the Leydig cell tumors of the testes were decreased. In hamsters, the incidence of adrenocortical adenomas were increased in the females. No other pertinent neoplastic changes were observed. In conclusion, the increases and decreases in benign neoplasms of hormone-sensitive tissues, appear to be the result of nutritionally/metabolism-induced modulation of the homeostasis in these 4 tissues in both species, and not the result of chronic glucose administration.

Bovine J blood-group activity in the lipids of erythrocytes of different age.

J-positive cattle erythrocytes were separated according to age by ultracentrifugation in a discontinuous density gradient of Ficoll-400 (Pharmacia) in isotonic buffer solution. A decrease in phospholipid and in cholesterol content with increasing age was detected. The J activity was found to increase markedly with increasing cell age. This fact supports the view that the in vivo transfer of the J determinant from plasma to erythrocytes is a rather slow process. It is suggested that the increasing J activity of older cells is probably due to a decrease of erythrocyte membrane bound phospholipids which inhibit the transfer of the J determinant to the red cells.

Distribution of the A blood group activity on lipid and nonlipid fractions of pig organs.

Lipids and protein-containing nonlipids of pig erythrocytes, serum and several organs were tested for A blood group activity. Both lipids and nonlipids of brain, myocardium, skeletal muscle and adipose tissue show no A activity. The way of distribution of A activity on lipids and nonlipids of other tissues and serum differs among individual pigs. With respect to the acquisition of A activity of erythrocytes in a postnatal period, it seems likely that the lipidic A substance Tor part of it containing the A determinant) is transferred from plasma to the erythrocyte membrane.

Transfer of bovine J blood-group activity to human erythrocytes in vitro.

THe bovine J determinant is transferred from a bovine serum nonlipid fraction to a human erythrocyte membrane lipid by an incubation procedure. The transferred J determinant is detected in the total lipids extracted from transformed human erythrocytes by an inhibition test in the bovine J system. It is also detected by cross-reacting human anti-A sera after treatment of human cells with papain prior or subsequent to transformation.

The transfer of J blood-group activity from J-containing bovine serum to J-negative erythrocytes.

The J blood-group activity of bovine serum is contained both in a lipid and in a nonlipid fraction. This is also true for calf serum. It demonstrated that the J determinant is transferred from a serum protein onto the erythrocyte membrane by incubation in vitro. Even though the donor of J activity is a lipid-free serum protein (probably a glycoprotein), the transferred J activity is detectable only in the lipid fraction of erythrocytes. Thus, the J determinant (probably a carbohydrate unit) must have been detached from a serum glycoprotein and transferred to a lipidic receptor (probably a glycosphingolipid) at the erythrocyte membrane. It is suggested than an enzyme system located in or at the erythrocyte membrane is responsible for the transfer of J substance. The transfer of J substance is inhibited by a polar lipid present in bovine serum.